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\DOI{10.5802/crchim.360}
\datereceived{2024-07-23}
\daterevised{2024-10-05}
\dateaccepted{2024-10-28}
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\dateposted{2024-12-03}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Two decades of fluorine chemistry in Cergy}

\alttitle{Deux D\'{e}cennies de Chimie du Fluor \`{a} Cergy}

\author{\firstname{Julien} \lastname{Pytkowicz}\CDRorcid{0000-0002-8545-3046}}
\address{CY Cergy Paris Universit\'{e}, CNRS, BioCIS UMR 8076, 95000
Cergy Pontoise, France}
\address{Universit\'{e} Paris-Saclay, CNRS, BioCIS UMR
8076, 91400 Orsay, France}
\email[J. Pytkowicz]{julien.pytkowicz@cyu.fr}

\author{\firstname{Gr\'{e}gory} \lastname{Chaume}\CDRorcid{0000-0001-7460-4876}}
\addressSameAs{1}{CY Cergy Paris Universit\'{e}, CNRS, BioCIS UMR 8076, 95000
Cergy Pontoise, France}
\addressSameAs{2}{Universit\'{e} Paris-Saclay, CNRS, BioCIS UMR
8076, 91400 Orsay, France}
\email[G. Chaume]{gregory.chaume@cyu.fr}

\author{\firstname{Nathalie} \lastname{Lensen}\CDRorcid{0000-0003-0321-0516}}
\addressSameAs{1}{CY Cergy Paris Universit\'{e}, CNRS, BioCIS UMR 8076, 95000
Cergy Pontoise, France}
\addressSameAs{2}{Universit\'{e} Paris-Saclay, CNRS, BioCIS UMR
8076, 91400 Orsay, France}
\email[N. Lensen]{nathalie.lensen@cyu.fr}

\author{\firstname{Evelyne} \lastname{Chelain}\CDRorcid{0000-0002-2163-5123}}
\addressSameAs{1}{CY Cergy Paris Universit\'{e}, CNRS, BioCIS UMR 8076, 95000
Cergy Pontoise, France}
\addressSameAs{2}{Universit\'{e} Paris-Saclay, CNRS, BioCIS UMR
8076, 91400 Orsay, France}
\email[E. Chelain]{Evelyne.chelain@cyu.fr}

\author{\firstname{Thierry} \lastname{Brigaud}\CDRorcid{0000-0003-0932-5971}\IsCorresp}
\addressSameAs{1}{CY Cergy Paris Universit\'{e}, CNRS, BioCIS UMR 8076, 95000
Cergy Pontoise, France}
\addressSameAs{2}{Universit\'{e} Paris-Saclay, CNRS, BioCIS UMR
8076, 91400 Orsay, France}
\email[T. Brigaud]{thierry.brigaud@cyu.fr}

\thanks{French Ministry of Education and Research, CY Cergy Paris
Universit\'{e}, CNRS, ANR, CY Initiative of Excellence (grant
``Investissements d'Avenir''), French Fluorine Network (GIS-FLUOR)}

\keywords{\kwd{Fluorinated chiral compounds}
\kwd{Fluorinated amino acids}
\kwd{Fluorinated peptides}
\kwd{Fluorinated chiral auxiliaries}}

\altkeywords{\kwd{Compos\'{e}s chiraux fluor\'{e}s}
\kwd{Aminoacides fluor\'{e}s}
\kwd{Peptides fluor\'{e}s}
\kwd{Auxiliaires chiraux fluor\'{e}s}}

\begin{abstract}
The development of chiral fluorinated biomolecules such as amino acids,
peptides and pseudopeptides is attracting considerable interest at the
frontiers of organic synthesis and biological chemistry. We propose
in this review an overview of our contributions to organofluorine
chemistry in connection with biological applications and also
asymmetric synthesis these past twenty years in Universit\'{e} de
Cergy-Pontoise (now CY Cergy Paris Universit\'{e}).
\end{abstract}

\begin{altabstract}
Le d\'{e}veloppement de biomol\'{e}cules fluor\'{e}es chirales telles
que les acides amin\'{e}s, les peptides et pseudopeptides suscite un
int\'{e}r\^{e}t consid\'{e}rable aux fronti\`{e}res de la synth\`{e}se
organique et de la chimie biologique. Nous proposons dans cette revue
un aper\c{c}u de nos contributions dans le domaine de la chimie des
compos\'{e}s organofluor\'{e}s pour des applications biologiques mais
aussi pour la synth\`{e}se asym\'{e}trique ces vingt derni\`{e}res
ann\'{e}es \`{a} l'Universit\'{e} de Cergy-Pontoise (maintenant CY
Cergy Paris Universit\'{e}).
\end{altabstract}

\shortrunauthors

\maketitle

\vspace*{.4pc}

\twocolumngrid
\end{noXML}

\section{Introduction}\label{sec1}

The introduction of fluorine atoms into biomolecules is now a
well-established strategy in drug development~\cite{1,2,3,4}. In
particular, the incorporation of fluorinated amino acids and
pseudopeptidic units into peptides is an outstanding tool for the
modulation of their biophysical properties such as local
hydrophobicity, membrane permeability~\cite{5} and metabolic
stability~\cite{6}. Moreover, fluorine atoms can serve as highly
sensitive probes for $^{19}$F NMR spectroscopy~\cite{7,8}. The
development of highly efficient syntheses of enantiopure fluorinated
amino acids and pseudopeptide units is an essential prerequisite for
their use in peptide chemistry~\cite{9,10,11,12} and we present herein
our main contributions in this area. Applications of the enantiopure
fluorinated chiral auxiliary Fox will also be reviewed. 

\begin{scheme*}
\includegraphics{sc01}
\vspace*{7pt}
\caption{\label{sch1}Synthesis of (${R}$)- and (${S}$)-trifluoroalaninol.}
\vspace*{-2pt}
\end{scheme*}

\section{Synthesis of chiral fluorinated amino compounds}\label{sec2}
\vspace*{-5pt}

\subsection{Amines, amino alcohols, diamines}\label{sec21}
\vspace*{-5pt}

Chiral amines and ${\upbeta}$-amino alcohols are very interesting
compounds for biological use and for the design of chiral ligands or
auxiliaries. This is why their asymmetric synthesis is a major topic of
organic synthesis. Considering the interesting properties provided by
fluorinated groups within biologically active molecules, our laboratory
was interested in the synthesis of trifluoro- or difluoromethylated
amines and amino alcohols in enantiopure form. One of the main
synthetic strategies to produce non-racemic chiral amines and amino
alcohols is the asymmetric Strecker reaction followed by the reduction
of the so formed nitrile group. In 2010, our group described two
efficient routes for the synthesis of both enantiomers of
trifluoroalaninol in enantiopure form~\cite{13}. Starting from
($R$)-phenylglycinol derived fluorinated oxazolidines or imines, the
first route relies on a Strecker type reaction whereas the more
straightforward second one uses ethyl trifluoropyruvate as fluorinated
starting material. This second more direct route involves, as a key
step, the reduction of chiral oxazolidines or imines derived from ethyl
trifluoropyruvate. The convenient separation of the two diastereomers
of the common intermediate amino diol makes it possible to obtain
trifluoroalaninols in enantiopure form in good yields. These two routes
use common and cheap reactants and can be scaled up to gram quantities
(Scheme~\ref{sch1}).

In order to provide simple and highly stereoselective access to
${\upbeta}$-alkyl ${\upbeta}$-trifluoromethyl ${\upbeta}$-amino
{alcohols, we decided to study addition reactions of organometallic
species to a chiral 2-hydroxymethyl fluorinated
oxazolidines~\cite{14}. These latter were obtained by chemoselective
NaBH}$_{4}$ reduction of oxazolidine derived from
trifluoropyruvate in very good yield as a 71:29 diastereomeric mixture.
Each diastereomer could be easily isolated by {chromatography} on silica
gel or by precipitation of the main diastereomer in pentane. The major
diastereomer of reduced oxazolidine was treated with an excess of
methyllithium at ${-}$78~\textdegree C in THF to give the amino diol
($R$) in 93\% yield and full diastereoselectivity. We showed
that the addition of methyllithium to the minor diastereoisomer gave
the same amino diol ($R$) in 80\% yield and 90\%
diastereoselectivity.


\begin{scheme*}
\includegraphics{sc02}
\caption{\label{sch2}Synthesis of ${\upbeta}$-trifluoromethyl ${\upbeta}$-amino 
alcohols.}
\vspace*{-5pt}
\end{scheme*}

We therefore considered that the same transition state should be
involved in the addition reaction of methyllithium to the two
diastereomers of reduced oxazolidines. This result was confirmed by the
fact that the addition of methyllithium to a diastereomeric mixture of
oxazolidines also gave the unique diastereomer ($R$) with excellent
diastereoselectivity. More generally, the addition of organometallic
species to fluorinated oxazolidines made it possible to obtain, after
cleavage of the phenylglycinol chain, quaternarized ${\upbeta}$-amino
alcohols in the ${\upbeta}$ position with good yields and excellent
diastereoselectivity (Scheme~\ref{sch2}).



\begin{scheme*}
\includegraphics{sc03}
\caption{\label{sch3}Synthesis of enantiopure
${\upalpha}$-trifluoromethyl diamines and amino alcohols.}
\vspace*{-5pt}
\end{scheme*}


Our group has also developed a straightforward synthetic route for the
synthesis of both enantiomers of various enantiopure diamines and amino
alcohols from trifluoromethyl ${\upalpha}$-amino nitriles as key
intermediates~\cite{15}. The Strecker-type reaction with TMSCN required
Lewis acid activation of fluorinated imines or oxazolidines, and the
reaction was promoted very efficiently under mild conditions with a
catalytic amount of Yb(OTf)$_{3}$. Despite the moderate
diastereoselectivity of the Strecker-type reaction, the high efficiency
of the chromatographic separation of each ${\upalpha}$-amino nitrile
diastereomer assisted by the ($R$)-phenylglycinol side chain allowed
the very convenient synthesis of enantiopure compounds
(Scheme~\ref{sch3}).


In 2005 we proposed a highly stereoselective method for the synthesis
of ${\upalpha}$-trifluoromethyl amines~\cite{16}. Indeed, we have shown
that organolithium and Grignard reagents add efficiently with very high
stereoselectivity (${>}$98\% de) to a trifluoroacetaldehyde hydrazone
derived from ($R$)-$N$-benzylphenylglycinol. The addition proceeds on
the \textit{Re} face of the chelated hydroxyhydrazone providing
quantitatively the ($R$)-${\upalpha}$-trifluoromethylated amine after
hydrogenolysis (Scheme~\ref{sch4}).

\begin{scheme*}
\includegraphics{sc04}
\caption{\label{sch4}Synthesis of enantiopure
${\upalpha}$-trifluoromethylamines.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc05}
\vspace*{5pt}
\caption{\label{sch5}Synthesis of enantiopure
${\upalpha}$-difluoromethylamines.}
\vspace*{-5pt}
\end{scheme*}


In 2008, our group reported a synthesis of enantiopure
${\upalpha}$-difluoromethylamines~\cite{17}. The silylated
difluoroenols formed in a first step can behave as electrophilic
reagents with amino alcohols, allowing the formation of oxazolidines.
The use of (${R}$)-phenylglycinol therefore made it possible to prepare
2-difluoromethyloxazolidines which proved to be good precursors of
enantiopure ${\upalpha}$-difluoromethylamines by lithium aluminium
hydride (LAH) reduction and oxidative cleavage of the intermediate
amino alcohols (Scheme~\ref{sch5}). The key 2-difluoromethyloxazolidine
intermediates are prepared in high yield from acylsilanes, treated
successively with trifluoromethyltrimethylsilane and the amine 
reagent.

\subsection{Amino acids}\label{sec22}


\subsubsection{Fluoroalkylamino acids} 


\paragraph{Synthesis of acyclic ${\upalpha}$-fluoroalkyl non
proteogenic amino acids}

Over the past 20 years, our group has devoted a great deal of effort to
developing a general methodology for accessing non-proteogenic
fluoroalkyl amino acids. These studies have been carried out for the
synthesis of non-proteinogenic ${\upalpha}$- or
${\upgamma}$-trifluoromethylated or ${\upalpha}$-difluoromethylated
amino acids, whether cyclic or linear. In most cases, the Strecker-type
reaction and the use of a chiral inducer such as the commercially
available phenylglycinol have been used for several reasons: 
\begin{itemize}
\item The Strecker-type reaction provides the nitrile function
precursor to the carboxylic function of the expected amino acids.
\item Phenylglycinol enables the expected enantiopure amino acids to be
synthesized.
\item It provides the nitrogen atom of the expected amino acid.
\item The phenylethanol group on the nitrogen atom can be easily
removed by acid hydrolysis or hydrogenolysis at the end of synthesis,
at the same time as hydrolysis of the nitrile function to carboxylic
acid. 
\item The source of fluorinated groups is commercially available and
therefore easily accessible.
\end{itemize}

\begin{scheme*}
\includegraphics{sc06}
\vspace*{5pt}
\caption{\label{sch6}Synthesis of enantiopure (${S}$) or
(${R}$)-${\upalpha}$-Tfm-allylglycine and
(${S}$)-${\upalpha}$-Tfm-norvaline.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc07}
\vspace*{2pt}
\caption{\label{sch7}Synthesis of enantiopure (${S}$) or
(${R}$)-${\upalpha}$-Tfm-aspartic acid and (${S}$) or
(${R}$)-${\upalpha}$-Tfm-serine.}
\vspace*{-5pt}
\end{scheme*}

In 2006, an allylation reaction on trifluoromethylated oxazolidine
obtained by condensation of trifluoropyruvate with ($R$)-phenylglycinol
enabled the construction of amino acid side chains and the synthesis of
enantiopure ${\upalpha}$-Tfm-${\upalpha}$-allylglycine and
${\upalpha}$-Tfm-norvaline thanks to possible separation of
diastereomers during the morpholinone formation step~\cite{18}. The
pure major diastereomer was then converted to the enantiopure
($S$)-${\upalpha}$-Tfm-norvaline via two hydrogenolysis steps
(Scheme~\ref{sch6}). 

In 2011, the Strecker-type strategy was implemented for the synthesis
of enantiopure ($S$)- and ($R$)-${\upalpha}$-Tfm-serine and
${\upalpha}$-Tfm-aspartic acid in good yields
(Scheme~\ref{sch7})~\cite{19}. The ethyl trifluoropyruvate based
oxazolidine was reduced and submitted to the Strecker-type reaction to
provide the corresponding hydroxymethyl amino nitriles. After
separation of each diastereomer and acidic hydrolysis, enantiopure
($S$)- and ($R$)-${\upalpha}$-Tfm-serine were obtained in good yields.
A similar strategy starting from the oxazolidine derived from ethyl
4,4,4-trifluoroacetoacetate provided enantiopure ($S$)- and
($R$)-${\upalpha}$-Tfm-aspartic acid in good yields
(Scheme~\ref{sch7})~\cite{19}.

Enantiopure ${\upalpha}$-Tfm-homoserine has also been obtained as a
ring-opening product during the synthesis of
2-trifluoromethyl-2-carboxyazetidine (Scheme~\ref{sch8})~\cite{20}. 

\begin{scheme*}
\includegraphics{sc08}
\vspace*{5pt}
\caption{\label{sch8}Synthesis of enantiopure ${\upalpha}$-Tfm-homoserine.}
\vspace*{-5pt}
\end{scheme*}


The laboratory was also interested in the synthesis of difluorinated
amino acids. This work was initiated in 2008, with the synthesis of
enantiopure ($S$) or ($R$)-${\upalpha}$-difluomethylalanine from the
condensation of acylsilanes and CF$_{3}$TMS, via the in-situ formation
of silyl ethers of difluoroenol. Condensation of this difluoroenol
silyl ether with the ($R$)-phenylglycinol enabled the preparation of
\mbox{diastereomerically} pure \unskip\break 2-difluoromethyl oxazolidines. A Strecker type
reaction followed by an oxidation led to chiral cyanodifluoroimines.
Acid hydrolysis gave access to the expected enantiopure ($S$)
or ($R$)-${\upalpha}$-difluoromethylalanine
(Scheme~\ref{sch9})~\cite{17}.

\begin{scheme*}
\includegraphics{sc09}
\vspace*{5pt}
\caption{\label{sch9}Synthesis of enantiopure (${S}$) or
(${R}$)-${\upalpha}$-difluoromethylalanine.}
\vspace*{-6pt}
\end{scheme*}

In 2023, we reported a synthesis of enantioenriched (${>}$90\% ee)
$N$-Fmoc-${\upbeta}$,${\upbeta}$-difluoroalanine employing
the Strecker's strategy from a chiral difluorinated aldimine. The
Strecker reaction produced two diastereomeric amino nitriles which were
separated. Subsequent selective hydrolysis reactions yielded the final
two enantiomers of difluoroalanine in good overall yield with an
enantiomeric excess superior to 90\% (Scheme~\ref{sch10})~\cite{21}.

\begin{scheme*}
\includegraphics{sc10}
\vspace*{5pt}
\caption{\label{sch10}Synthesis of enantiopure (${S}$) or
(${R}$)-${N}$-Fmoc-${\upbeta}$,${\upbeta}$-difluoroalanine.}
\vspace*{-5pt}
\end{scheme*}

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc11}
\vspace*{5pt}
\caption{\label{sch11}General pathway for the synthesis of enantiopure
cyclic (${R}$) or (${S}$)-${\upalpha}$-Tfm-${N}$-protected amino
acids.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc12}
\caption{\label{sch12}Synthesis of enantiopure
${N}$-protected-${\upalpha}$-Tfm-aziridine 2-carboxylic acid.}
\vspace*{-5pt}
\end{scheme*}

\paragraph{Synthesis of cyclic ${\upalpha}$-fluoroalkyl non proteogenic
amino acids} 

Another area of research was the synthesis of more constrained cyclic
${\upalpha}$-trifluoromethylated amino acids. The synthesis routes were
the same as those used for acyclic ones. The main strategy is based on
the condensation of various commercially available trifluoromethylated
ketoesters with enantiopure ($R$)-phenylglycinol. The corresponding
chiral oxazolidines were obtained in good yields. Through various
chemical transformations, monocyclic and bicyclic intermediates were
synthesized. To introduce the nitrile group, {precursor} of the carboxy
group, a Strecker-type reaction assisted by boron trifluoride Lewis
acid on these chiral cyclic intermediates was chosen. While these
Strecker type reactions proceeded in very high yields, only modest
diastereoselectivities were probably obtained due to the passage
through an iminium intermediate. Nevertheless, the diastereomeric
mixture was resolved via silica gel separation to obtain
enantiomerically pure batches of each amino nitrile. Once these
aminonitriles synthesized, it was necessary to find the conditions for
nitrile hydrolysis and protection of the amine function
(Scheme~\ref{sch11}). 


In the case of aziridine, strong acid conditions led to the aziridine
ring opening and formation of a complex mixture. Nevertheless, after
mesylation of the primary alcohol function of oxazolidine followed by a
Strecker-type reaction and separation of the diastereomer, the expected
($R$,$S$)-$N$-protected ${\upalpha}$-trifluoromethyl
aziridine-2-carboxylic acid (${\upalpha}$-TfmAzy) was obtained after
basic treatment (Scheme~\ref{sch12})~\cite{22}.

Another strategy to access these enantiopure ${\upalpha}$-TfmAzy was
via the intramolecular cyclization of enantiopure ($S$) or
($R$)-${\upalpha}$-trifluoromethyl serine derivatives already
synthesized in the laboratory (Scheme~\ref{sch13})~\cite{22}. 

\begin{scheme*}
\includegraphics{sc13}
\caption{\label{sch13}Synthesis of enantiopure
${N}$-Bn-${\upalpha}$-Tfm-aziridine 2-benzyloxycarbonyl.}
\end{scheme*}

For ${\upalpha}$-trifluorometylated azetidine, the use of strong acidic
conditions cleaved the azetidine ring to give
${\upalpha}$-trifluoromethylated homoserine (see Scheme~\ref{sch8}).
Milder conditions were found, and the synthesis via an intermediate
bicyclic compound followed by hydrogenolysis gave access to the
enantiomerically pure (2$R$)-2-trifluoromethyl-2-carboxyazetidine
(Scheme~\ref{sch14})~\cite{20}. 

\begin{scheme*}
\includegraphics{sc14}
\caption{\label{sch14}Synthesis of enantiopure
(2${R}$)-2-trifluoromethyl-2-carboxyazetidine.}
\vspace*{-5pt}
\end{scheme*}

The synthesis of constrained ${\upalpha}$-trifluoromethyla\-ted or
5-Tfm-proline has been a major research focus in the laboratory. Three
synthetic routes have been developed. The first one was based on an
allylation reaction on chiral 2-trifluoromethyloxazolidines derived
from ethyl trifluoropyruvate to build up the amino acid side chain, the
second one used a Strecker-type reaction to introduce the carboxy
function, and finally the third one used Claisen-type \mbox{reactions}.

Starting from the Tfm-allylmorpholinone intermediate resulting from the
pseudoproline allylation reaction, several synthetic routes are
possible. 

{\smallskip\noindent}
\textbullet~~Synthetic Route 1.


Hydroboration followed by oxidation led to the formation of the
hydroxymorpholinone which then undergoes a cyclization reaction via
mesylation. The bicycle formed then undergoes a hydrogenolysis
reaction, giving access to the two enantiomers of
${\upalpha}$-Tfm-proline~\cite{18}.

{\smallskip\noindent}
\textbullet~~Synthetic Route 2.

An iodocyclization reaction on Tfm-allylmorpholinone led to a bicyclic
iodo compound, which via a hydrogenolysis reaction, also led to the
formation of enantiopure ${\upalpha}$-Tfm-proline on a gram scale. The
bicyclic iodo compound, by elimination of the iodine in a basic medium
followed by a dihydroxylation reaction, gave access to the
${\upalpha}$-Tfm-dihydroxyproline~\cite{23}.

{\smallskip\noindent}
\textbullet~~Synthetic Route 3.

A hydroboration reaction on CF$_{3}$-allylmorpholi\-none followed by two
oxidation reactions, saponification and deprotection of the amine
function yields enantiopure ${\upalpha}$-Tfm-pyroglutamic acid
(Scheme~\ref{sch15})~\cite{24}. 

\begin{scheme*}
\includegraphics{sc15}
\vspace*{5pt}
\caption{\label{sch15}Synthesis of enantiopure
${\upalpha}$-Tfm-proline, ${\upalpha}$-Tfm-dihydroxyproline and
${\upalpha}$-Tfm-pyroglutamic acid.}
\vspace*{-5pt}
\end{scheme*}

For the synthesis of the enantiopure 5-Tfm-proline, an interesting
constrained non-natural amino acid, our laboratory devised the
following synthetic scheme (Scheme~\ref{sch16})~\cite{25}. The first
step was the condensation of fluoral with (${R}$)-phenylglycinol,
yielding the CF$_{3}$-oxazolidine of two inseparable diastereomers.
Addition of an allyllithium reagent gave a mixture of the expected enol
ether again in an inseparable mixture of diastereomers. Treatment under
acidic conditions led to a fluorinated oxazolopyrrolidine also in a
mixture of diastereomers. An enantiomerically pure oxazolopyrrolidine
intermediate was obtained after efficient kinetic resolution by
addition of 0.7 equivalent PhMgBr based on selective consumption of the
undesirable minor diastereomer. A classical Strecker type reaction on
this bicyclic intermediate followed by a separation of the diastereomer
led by an acid hydrolysis step to a mixture of diastereomer. Basic
conditions on a pure diastereomer led after hydrogenolysis the
enantiopure ($2{S},5{R}$)-5-trifluoromethylproline. The synthetic
potential of this methodology was demonstrated by the scalable
synthesis of the highly valuable enantiopure
($2{S},5{R}$)-5-trifluoromethylproline\break
(Scheme~\ref{sch16})~\cite{25}.


\begin{scheme*}
\includegraphics{sc16}
\caption{\label{sch16}Synthesis of enantiopure
(2${S}$,5${R}$)-5-trifluoromethylproline.}
\vspace*{-3pt}
\end{scheme*}

In 2021, we described a new straightforward synthesis of enantiopure
5-(${R}$)-and 5-(${S}$)-trifluoromethyl proline via a shorter multistep
procedure avoiding tedious diastereomers separation
(Scheme~\ref{sch17})~\cite{26}. The key steps are a Ruppert--Prakash
reagent addition on the properly protected L-pyroglutamic acid followed
by an elimination reaction and a diastereoselective reduction occurring
on the less hindered face of the imine to provide the \textit{cis}
isomers. In order to obtain the \textit{trans} isomer, the hydride
reduction of the imine was investigated and the best results were
obtained with NaBH$_{3}$CN giving access to the \textit{trans} benzyl
ester of 5-CF$_{3}$-proline after chromatographic separation on silica
gel. Finally, an hydrogenolysis of the benzyl group provided
\textit{trans} 5-Tfm-proline (${S}$) and the \textit{cis} 5-Tfm-proline
(${R}$) (Scheme~\ref{sch17})~\cite{26}.

\begin{scheme*}
\includegraphics{sc17}
\caption{\label{sch17}Synthesis of enantiopure 5-trifluoromethylproline.}
\vspace*{-5pt}
\end{scheme*}

We also reported, in 2021, a new simple and scalable synthesis of the
two enantiomers of ${\upalpha}$-Tfm-proline~\cite{27}. In order to
generalize to higher cyclic proline analogues, the synthesis of the two
enantiomers of ${\upalpha}$-Tfm-pipecolic acid was also reported.
Enantiopure ${\upalpha}$-Tfm-proline and ${\upalpha}$-Tfm-pipecolic
acid were synthesized starting from commercially available diesters and
ethyl trifluoroacetate via Claisen condensation (Scheme~\ref{sch18}).

\begin{scheme*}
\includegraphics{sc18}
\caption{\label{sch18}Synthesis of a common intermediate for
${\upalpha}$-Tfm-proline and ${\upalpha}$-Tfm-pipecolic acid.}
\end{scheme*}

At this stage, two different pathways were used for the proline and the
pipecolic acid series. For the proline route, the oxazolidine was
reduced with NaBH$_{4}$/CaCl$_{2}$ to give the alcohol. Its
\mbox{iodocyclization} gave access to the Tfm-oxazolopyrrolidines. For the
pipecolic route, a simple cyclization gave a lactam which was then
reduced to give Tfm-oxazolopiperidines (Scheme~\ref{sch19}).

\begin{scheme*}
\vspace*{4pt}
\includegraphics{sc19}
\vspace*{6pt}
\caption{\label{sch19}Synthesis of Tfm-oxazolopyrrolidines and
Tfm-oxazolopiperidines intermediates.}
\vspace*{5pt}
\end{scheme*}

A Strecker type reaction on chiral Tfm-oxazolo-pyrrolidine and
piperidine intermediates yielded the corresponding nitriles, precursors
of enantiopure ($R$) and ($S$) ${\upalpha}$-Tfm-proline and
${\upalpha}$-Tfm-pipecolic acid (Scheme~\ref{sch20})~\cite{27}.

\begin{scheme*}
\includegraphics{sc20}
\vspace*{6pt}
\caption{\label{sch20}Synthesis of enantiopure ($R$) and ($S$)
${\upalpha}$-Tfm-proline and ${\upalpha}$-Tfm-pipecolic acid.}
\vspace*{-2pt}
\end{scheme*}


\subsubsection{Pseudoprolines}

Fluorinated oxazolidines are conveniently synthesized by condensation
reaction of serine esters and hemiacetal trifluoroacetaldehyde or
trifluoroacetone~\cite{28}. These oxazolidines are stabilized towards
ring opening by the electron-attracting character of the
trifluoromethyl group and can be considered as pseudoprolines
(${\rmPsi}$pro) (Scheme~\ref{sch21}). Each diastereomer can be isolated
and they are not in equilibrium in neutral conditions. Despite the
deactivation of the nitrogen atom, these oxazolidines can be
efficiently $N$-acylated to give stable pseudoproline-type structures
as \textit{cis}/\textit{trans} amide conformers. Conformational study
of Tfm-pseudoprolines and \textit{cis}/\textit{trans} isomerization are
the subject of a following part of this review. For future applications
in peptide chemistry, pseudoprolines are hydrolytically stable
authentic proline surrogates, compatible with the SPPS Fmoc/$t$-Bu
strategy.

\begin{scheme*}
\includegraphics{sc21}
\caption{\label{sch21}Synthesis of Tfm${\rmPsi}$pro esters.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc22}
\caption{\label{sch22}Synthesis of CF$_{2}$H-${\rmPsi}$pro esters.}
\vspace*{-4pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc23}
\caption{\label{sch23}Early and late-stage access of TFM and TFM
containing peptides.}
\vspace*{-6pt}
\end{scheme*}


Similarly, in 2019, original enantiopure (${S}$,${S}$) and
(${R}$,${S}$)-CF$_{2}$H-pseudoprolines were synthesized as stable
5-substituted proline substitutes that could be incorporated into a
peptide chain (Scheme~\ref{sch22})~\cite{29}. 


\subsubsection{SCF$_3$-amino acids}
\vspace*{-4pt}

Among the fluorinated noncanonical amino acids, L-trifluoromethionine
(TFM) and L-(S-trifluoromethyl)cysteine (Tfm-Cys), fluorinated
analogues of methionine and cysteine, are of particular interest
because of their ability to locally increase the hydrophobicity of
peptides~\cite{30}. In 2017, our group reported the synthesis of
\textit{tert}-butoxycarbonyl/benzyl-protected TFM and Tfm-Cys using
the cheap and user-friendly radical trifluoromethylation reagent
previously developed by Langlois and co-workers~\cite{31}. This
early-stage strategy allowed us to obtain the fluorinated noncanonical
amino acids on gram scales, ready to use in liquid phase peptide
synthesis (Scheme~\ref{sch23})~\cite{32}. In addition to this strategy,
we reported a late-stage strategy inspired by the pioneer work of
Seebach and Togni based on the electrophilic trifluoromethylation of a
dimeric disulfide precursor. After the reduction of the precursor with
tris-(2-carboxyethyl)phosphine (TCEP), electrophilic
trifluoromethylation gave the expected peptides in low to moderate
yield after purification, demonstrating that this strategy, already
known for Tfm-Cys, was transposable to the synthesis of TFM-containing
peptides.



Recently, we extended a methodology developed for the
trifluoromethylthiolation of aromatic substrates~\cite{33}, to the
synthesis of Fmoc-protected trifluoromethylthiolated tyrosine
(CF$_{3}$S-Tyr) and tryptophan (CF$_{3}$S-Trp) analogues on a gram
scale (77--93\% yield)~\cite{33}. The developed conditions are also
efficient on tryptamine, tyramine and L-DOPA (Scheme~\ref{sch24}). 

\begin{scheme*}
\includegraphics{sc24}
\vspace*{2pt}
\caption{\label{sch24}Trifluoromethylthiolation of substrates of interest.}
\vspace*{-4pt}
\end{scheme*}

In addition to the use of Fmoc-protected trifluoromethylthiolated
tryptophanes and tyrosines in classical Solid Phase Peptide Synthesis
(SPPS), the developed methodology was successfully applied to the
late-stage regioselective trifluoromethylthiolation of Trp residues in
short peptides. To prove the concept and explore the regioselectivity
of the reaction, Fmoc-(CF$_{3}$S)-Tyr and Fmoc-(CF$_{3}$S)-Trp were
incorporated into the endomorphin-1 chain (EM-1) and into model
tripeptides obtained by SPPS (Scheme~\ref{sch25})~\cite{33}. 

\begin{scheme*}
\includegraphics{sc25}
\vspace*{2pt}
\caption{\label{sch25}Trifluoromethylthiolation of short peptides.}
\vspace*{-2pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc26}
\vspace*{2pt}
\caption{\label{sch26}Diastereoselective alkylation of
\textit{trans}-Fox derived amide enolates.}
\vspace*{-4pt}
\end{scheme*}

\section{Applications}


\subsection{Asymmetric synthesis} 

In 2006, we described the use of the enantiopure trifluoromethylated
oxazolidine derived from phenylglycinol (\textit{trans}-Fox) as a very
efficient chiral auxiliary~\cite{34}. The starting point of this study
was the excellent configurational stability of
2-trifluoromethyloaxazolidines derived from enantiopure aminoalcohols.
Once acylated, unlike their non-fluorinated counterparts, the two
positions of the fluorinated oxazolidines did not epimerize neither in
acidic nor basic conditions. This property allowed us to isolate the
\textit{trans} substrates derived from the acylation of the
(4${R}$)-2-trifluoromethyl-4-phenyl-1,3-oxazolidine
(\textit{trans}-\textbf{Fox amide}, Scheme~\ref{sch26}). We
hypothesized that the configurationally stable trifluoromethyl group
could be a very efficient chiral inducer due to its steric hindrance
comparable to the \textit{iso}-propyl or \textit{tert}-butyl group.
Indeed, the alkylation of amide enolates derived from various alkylated
substrates proved to be completely diastereoselective (${>}$99\%
\textit{de}) when performed in THF at ${-}$78~\textdegree C using various
alkylated electrophiles ($R_{2} = \mathrm{Me}$, Et, Allyl, Bn, $i$-Pr,
$t$-Bu). These results highlight the great potential of this
(${R}$)-phenylglycinol-based fluorinated oxazolidine as chiral
auxiliary for asymmetric alkylation reactions and good alternative to
the classical Evan's oxazolidinones. Removal of the chiral auxiliary
could be performed using reductive conditions. Treatment of the
alkylated product with LiAlH$_{4}$ afforded the corresponding aldehydes
together with the deacylated oxazolidine that could be further
recovered and reused. Enantiopure alcohols or carboxylic acids could be
obtained from the aldehydes using a reductive or oxidative sequential
two steps procedure~\cite{34}.

Disappointing results obtained for the alkylation reactions of the
\textit{cis}-Fox amides, confirmed that a
pseudo-\textit{C2}-symmetry of the oxazolidine is mandatory to
reach high levels of diastereoselectivity. The origin of the excellent
stereocontrol was investigated by an experimental and theoretical (DFT)
study~\cite{35}. We showed that, in the presence of the
\textit{trans}-Fox auxiliary, both F${\cdots}$Na$^+$ and
${\uppi}{\cdots}\mathrm{Na}^+$ interactions compete to give the
same diastereomer through \textit{Re} face alkylation of the enolate. A
5.5~kcal${\cdot}$mol$^{-1}$ energy difference found between the
\textit{Re} face and the \textit{Si} face attack transition states is
consistent with the complete diastereoselectivity that has been
experimentally achieved (Figure~\ref{fig1}). In the case of the
\textit{cis}-Fox chiral auxiliary, the competition between the
F${\cdots}$Na$^+$ and ${\uppi}{\cdots}$Na$^+$ led to nearly isoenergetic
(0.3~kcal${\cdot}$mol$^{-1}$) \textit{Re} and \textit{Si} face attacks
which are in good agreement with the very low diastereoselectivity
experimentally observed.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Theoretical \textit{Re} face (TS1) and \textit{Si}
face (TS2) transition states with \textit{trans}-Fox chiral
auxiliary.}
\vspace*{2pt}
\end{figure*}

\begin{scheme*}
\includegraphics{sc27}
\vspace*{5pt}
\caption{\label{sch27}Diastereoselective alkylation of
\textit{trans}-Fox derived ${\upbeta}$-amino substrates.}
\vspace*{-1pt}
\end{scheme*}

The alkylation reaction of \textit{trans}-Fox amides with various
halogenated electrophiles was later extended to ${\upbeta}$-amino
substrates~\cite{36}. The alkylation with benzyl bromide, allyl
bromide, and ethyl iodide was performed in good yield and with complete
diastereoselectivity. Interestingly, the reaction with more hindered
halogenated compounds such as isobutyl iodide and isopropyl iodide
giving rise to ${\upbeta}$-2-homoleucine and ${\upbeta}$-2-homovaline
precursors were also completely diastereoselective. As previously
described, removal of the chiral auxiliary was achieved in a two-step
procedure involving a LiAlH$_{4}$ reduction of the alkylation product
followed by the oxidation or the NaBH$_{4}$ reduction of the
intermediate amino aldehyde (Scheme~\ref{sch27}).


In the course of previous studies, ${\upalpha}$-hydroxylated or
${\upalpha}$-hydroperoxylated amide were occasionally observed as side
products of the alkylation reactions. We hypothesized that these
oxygenated compounds probably resulted from the enolate oxidation by
residual molecular oxygen. Submitted to a stream of bubbling molecular
oxygen at ${-}$78~\textdegree C, \textit{trans}-Fox amide enolate led to
the corresponding hydroperoxide with good diastereoselectivity. When
the hydroperoxide is reduced in situ with triethylphosphite,
${\upalpha}$-hydroxy amides were obtained in good yield and with
excellent diastereoselectivity~\cite{37}. After benzyl protection of
the newly formed alcohol, LiAlH$_{4}$ reduction followed by subsequent
sodium chlorite oxidation or NaBH$_{4}$ reduction gave the
corresponding enantiopure carboxylic acids and alcohols
(Scheme~\ref{sch28}).

\begin{scheme*}
\includegraphics{sc28}
\caption{\label{sch28}Diastereoselective ${\upalpha}$-hydroxylation of
\textit{trans}-Fox derived amide enolates.}
\vspace*{-2pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc29}
\caption{\label{sch29}CIDR of \textit{trans}-Fox and diastereoselective
${\upalpha}$-fluoration of \textit{trans}-Fox amides.}
\vspace*{-2pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc30}
\caption{\label{sch30}Diastereoselective aldol reaction of
\textit{trans}-Fox amide.}
\vspace*{-4pt}
\end{scheme*}

In 2013, our group reported a highly efficient crystallization-induced
dynamic resolution (CIDR) of \textit{trans}-Fox. Taking advantage of
the rapid equilibrium between \textit{cis} and \textit{trans}
conformers of fluorinated oxazolidines in acidic medium, pure
\textit{para}-toluene sulfonic acid salt of \textit{trans}-Fox could be
crystallized from a mixture of \textit{cis--trans} diastereomers in
ethyl acetate. This dynamic resolution allowed us to prepare the
\textit{trans}-Fox chiral auxiliary on multigram scale avoiding
subsequent tedious separation of \textit{cis} and \textit{trans}
amides~\cite{38}. The electrophilic fluorination of \textit{trans}-Fox
amides with ${N}$-fluorobenzenesulfonimide (NFSI) proceeded smoothly at
low temperature to give the ${\upalpha}$-fluoro amides with excellent
diastereoselectivity. Corresponding ${\upalpha}$-fluoro carboxylic
acids and ${\upbeta}$-fluoro alcohols could be obtained using the
previously described procedure (Scheme~\ref{sch29}).

\begin{scheme*}
\includegraphics{sc31}
\vspace*{5pt}
\caption{\label{sch31}Selected examples of solution phase or SPPS
synthesis of peptide incorporating difluoroalanine and SCF$_{3}$ group
containing amino acids.}
\end{scheme*}

Aldol reactions of sodium amide enolates derived from
\textit{trans}-Fox chiral auxiliary occurred in good yields but
moderate \textit{anti} diastereoselectivity. \textit{Syn}
diastereoselectivity was obtained using boron enolates. After removal,
the Fox chiral auxiliary is very conveniently and efficiently recovered
in basic conditions (Scheme~\ref{sch30})~\cite{39}.
\vspace*{-4pt}

\subsection{Incorporation in peptides and modulation of the biophysical
and biological properties of peptides} 
\vspace*{-4pt}


\subsubsection{Incorporation in peptides}

The incorporation of ${\upalpha}$-amino acids bearing a fluorinated
side chain in peptides does not present any particular difficulties
when the fluorinated group is not deactivating too much the amino group
of the amino acid. This is the case for difluoroalanine and SCF$_{3}$
group containing amino acids (Scheme~\ref{sch31}). Standard peptide
coupling reaction conditions can be used for solution phase or SPPS
incorporation of these amino acids in peptides~\cite{21,32,33}. 


The peptide coupling reactions at the $C$-terminal position of
${\upalpha}$-fluoroalkyl-${\upalpha}$-amino acids, 5-Tfm-prolines and
Tfm-pseudoprolines can also be achieved in good yields by standard
procedures (Scheme~\ref{sch32})~\cite{22,27,40,41,42}. It should be
noticed that because of the strong deactivation of the nitrogen atom of
the ${\upalpha}$-trifluoromethyl-${\upalpha}$-amino acids, the
protection of its amino group is not required.

\begin{scheme*}
\includegraphics{sc32}
\caption{\label{sch32}Selected examples of \textit{C}-terminal position
coupling peptides with solution phase or SPPS.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc33}
\caption{\label{sch33}Selected examples of ${N}$-terminal position
peptide coupling reactions.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc34}
\vspace*{6pt}
\caption{\label{sch34}Selected examples of peptides incorporating
${\upalpha}$-Tfm-alanine and 5-Tfm-proline synthesized by SPPS.}
\vspace*{-3pt}
\end{scheme*}

However, when fluorine atoms are located at the ${\upbeta}$-position of
the amino group of the amino acid, their strong electron-withdrawing
effect dramatically decreases the nucleophilicity of the nitrogen atom
and specific activation methods are generally required. The peptide
coupling reactions at the ${N}$-terminal position of
${\upalpha}$-Tfm-amino acids and especially the hindered quaternarized
ones, required the activation of the amino acid as a mixed {anhydride} or
an acyl halide (Scheme~\ref{sch33}). The mixed \mbox{anhydride} was prepared by
reaction of the amino acid with isobutyl chloroformate (IBCF) in the
presence of ${N}$-methylmorpholine (NMM). The amino acyl chlorides were
conveniently prepared by treatment of the amino acid with an excess of
thionyl chloride and evaporation. The coupling reaction of Fmoc-amino
acid chlorides were conveniently achieved at the ${N}$-terminal
position of ${\upalpha}$-Tfm-alanine~\cite{43},
${\upalpha}$-Tfm-proline~\cite{26}, CF$_{3}$- and
CF$_{2}$H-pseudoprolines~\cite{26,29,42,44}. It should be mentioned
that the ${N}$-terminal position coupling reaction on
CF$_{3}$-pseudoprolines requires the use of amino acids chlorides
without bases. In these conditions, the amide bond formation is
generally associated with an epimerization of the chiral center bearing
the trifluoromethyl group through a dynamic kinetic resolution
process~\cite{42}. Starting from \textit{cis} or \textit{trans}
CF$_{3}$-pseudoprolines, the only \textit{cis} CF$_{3}$-pseudoprolines
dipeptides are obtained. In the CF$_{2}$H-pseudoproline series, the
coupling \mbox{reaction} can be performed in the presence of pyridine and the
difluorinated pseudoprolines are obtained in good yield without
epimerization. Several examples of ${N}$-terminal position peptide
coupling conditions are presented in Scheme~\ref{sch33}. 

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Hydrophobic contribution of fluorinated amino
acids compared to their non-fluorinated analogues using the
Chromatographic Hydrophobic Indexes (CHI) method~\cite{21,30,33}.}
\end{figure*}

The direct incorporation of ${\upalpha}$-Tfm-AAs
(${\upalpha}$-trifluoromethyl amino acids) in the middle of a peptide
chain is still a challenge and all attempts to perform on resin the
coupling of an amino acid at the $N$-terminal position of the
deactivated fluorinated amino acid failed so far. The alternative
strategy implemented for the incorporation of such amino acid in long
peptides by SPPS is the incorporation of Fmoc-protected dipeptides
building blocks resulting from the solution phase coupling
(Scheme~\ref{sch34})~\cite{43,45}. According to this strategy, numerous
fluorinated labeled peptides of the peptaibol series, such as
Harzianin~\cite{45}, have been prepared notably for $^{19}$F NMR
studies of interactions with membranes. Interestingly, the 5-CF$_3$
proline can be conveniently incorporated in peptides by SPPS~\cite{26}.
\vspace*{-3pt}


\subsubsection{Hydrophobicity modulation and biological applications} 

The modulation of the hydrophobicity of peptides by the incorporation
of fluorinated amino acids is mostly reported and well documented in
the ${\upgamma}$- or ${\upomega}$-fluoro substituted series.
Generally, the monofluorination lowers the hydrophobicity compared to
the non-fluorinated analogues while the di- and trifluorination
increase it. Because of their difficult synthesis, the hydrophobicity
modulation assessment of peptides incorporating
${\upalpha}$-fluoroalkyl chains has been investigated only recently.
The approach we adopted consists in the HPLC Hydrophobic Indexes (CHI)
determination of peptides. According to this method, we measured the
CHI of peptides incorporating a ${\upalpha}$-fluoroalkyl amino acid by
comparison with their non-fluorinated analogues~\cite{30}. This method
allows an accurate assessment of the local hydrophobic contribution of
the fluorinated side chain compared to an aliphatic side chain
(Figure~\ref{fig2}). Di- and trifluorinated amino acids, such as
difluoroalanine (DfAla) and trifluoromethylalanine (Tfm-Ala), provided
an increased local hydrophobicity compared to alanine and
aminoisobutyric acid (Aib). The hydrophobicity index shift dCHI
($\mathrm{CHI}_{\mathrm{DfAla}} - \mathrm{CHI}_{\mathrm{Ala}} = 9.9$)
constitutes a quantitative assessment of the increased hydrophobic
contribution of the CF$_{2}$H substituent compared to the methyl
group of alanine~\cite{21}. It is very \mbox{interesting} to note that
despite its smaller van der Waals volume, the CF$_{2}$H group provides
nearly the same hydrophobic contribution than the side chain of
isoleucine (Ile), which is known to be the most hydrophobic aliphatic
amino acid ($\mathrm{CHI}_{\mathrm{DfAla}} - \mathrm{CH}_{\mathrm{IIle}} =
0.2$). Similarly, trifluoromethylalanine provides an increased
hydrophobicity compared to Aib
($\mathrm{CHI}_{\mathrm{TfmAla}} - \mathrm{CH}_{\mathrm{Aib}} = 6.7$)
(Figure~\ref{fig2})~\cite{30}. SCF$_3$ group containing amino acids are
providing a dramatic increase of hydrophobicity~\cite{30,33}.


This trend has been applied by us for the design of small hydrophobic
peptides for ${\upbeta}$-amyloid aggregation inhibition~\cite{46}. As
hydrophobic amino acids and/or the ${\upalpha}$-aminoisobutyric acid
(Aib) are known to act as ${\upbeta}$-sheet breakers, the
H-Ala--Aib--Leu-OH and H-Ala--($R$)-Tfm-Ala--Leu-OH peptides were
investigated as inhibitors of A${\upbeta}$42 aggregation. These
peptides were evaluated by the ThioflavinT (ThT) binding assay and both
showed an inhibition of the kinetics of fibril formation at high
concentration. The Tfm group containing peptide gave significantly
better inhibition results because of its increased hydrophobicity and
then a good affinity with the hydrophobic region of A${\upbeta}$.

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig03}
\vspace*{4pt}
\caption{\label{fig3}PAMPA method for measuring the increased passive
diffusion of ${\upalpha}$-Tfm-AAs containing short peptides through
membranes~\cite{47}.}
\end{figure*}

Another method reported in the literature for the hydrophobicity
evaluation of fluorinated peptides is the Parallel Artificial Membrane
Permeability assay (PAMPA)~\cite{47}. This method has been implemented
to investigate the ability of fluorinated analogues of the PLG peptide
(MIF-1) to cross the blood brain barrier (BBB). The fluorinated
analogue of PLG with an ${\upalpha}$-trifluoromethylproline in place of
the proline showed a better analgesic activity~\cite{48,49}. This
improved biological activity has been related to an increased
hydrophobicity and ability to cross the BBB through passive diffusion
as shown by PAMPA (Figure~\ref{fig3})~\cite{47}. 

\subsubsection{Increased resistance towards proteases and protease
inhibitor design} 

The modulation of the resistance of peptides towards proteolysis have
been mainly investigated with amino acids carrying fluorine atoms at
the ${\upgamma}$ or ${\upomega}$ position of the side chain~\cite{50}.
Once again, because of their difficult synthesis and incorporation in
peptides, ${\upalpha}$-fluoroalkyl amino acids have rarely been
studied. After the pioneer works by Burger and Koksch~\cite{51}, we
recently revisited the high potential of these amino acids to
circumvent peptides proteolysis. Pepsin is generally cleaving peptides
at the $C$-terminus of a phenylalanine residue (Phe). By incorporating
an ${\upalpha}$-Tfm-Ala adjacent to the Phe carboxylic group of the
peptide \textbf{A} (Figure~\ref{fig4}), the hydrolysis was almost
inhibited and a slow cleavage occurred at another position~\cite{52}. A
similar peptide incorporating an arginine (Arg) (peptide \textbf{B} in
Figure~\ref{fig4}) has been used as a fluorinated tag in a 3-FABS
$^{19}$F NMR based activity assay to probe the IC$_{50}$ of
a trypsin inhibitor~\cite{52}. Very recently we incorporated ($R$)-
and ($S$)-${\upalpha}$-Tfm-Ala as well as ($R$)- and ($S$)-TFM
(trifluoromethionine) in Pepstatin A based \mbox{inhibitors} of Cathepsin D
and Pepsin (peptides \textbf{C}, \textbf{D}, \textbf{E} and \textbf{F} in
Figure~\ref{fig4})~\cite{53}. The inhibitory potency of these Pepstatin
analogues was measured towards isolated Cathepsin D and Pepsin using a
classical FRET based assay. Both peptides presented nanomolar
IC$_{50}$. However, they were slightly less active and
selective than Pepstatin. The ($R$)-TFM based analog of Pepstatin
\textbf{A} ({peptide \textbf{E}, Figure~\ref{fig4}) returned a
sub-nanomolar IC$_{50}$ against CD and an increased
selectivity. Molecular Docking experiments clearly demonstrated the
stabilization of the inhibitor \textbf{E} in the catalytic pocket of
Cathepsin D by strong hydrophobic interactions due to the long and
flexible SCF$_3$ containing side chain.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Tfm-Ala-containing peptides for increased
resistance towards proteases and proteases inhibitors design.}
\vspace*{2pt}
\end{figure*}

Recently we also reported that a fluorinated polyproline PPII helix
incorporating trifluoromethyl pseudoprolines exhibits a strong
resistance in Pronase since no degradation was observed after two
days~\cite{54}.


\subsection{Conformational aspects of
${\alpha}$-Fluoroalkyl-$\alpha$-amino acids and their peptides}

\subsubsection{Cyclic $\alpha$-Fluoroalkyl-$\alpha$-amino acids}

Because of its cyclic structure, the proline residue is recognized to
play a unique and crucial role in the peptide backbone conformation and
biological properties. When included into a peptide, the {pyrrolidine}
ring cannot act as a hydrogen bond donor and restrains the ${\phi}$
dihedral angle to about ${-}$60\textdegree~(Figure~\ref{fig5}). The
Xaa--Pro peptide bond is characterized by a small free energy
difference ($\rmDelta G^{{\circ}}_{\mathrm{tc}}$) between the \textit{trans}
and the \textit{cis} amide bond conformers, combined with a high
activation energy (${\rmDelta}G^{\#}_{\mathrm{tc}}$) for the
\textit{cis}--{\textit{trans} isomerization. This isomerization is
considered to play a key role in regulating many important biological
processes in proline containing peptides and proteins, including
protein folding.

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}General features of proline residue when
incorporated into peptides.}
\end{figure*}

Many proline analogues have been reported in the literature to control
the \textit{cis}}--{\textit{trans} isomerization of the
peptidyl--prolyl amide bond. Our group investigated the ability of our
CF$_{3}$- and CF$_{2}$H-substituted prolines and pseudoprolines to
serve as attractive tools for the control of the peptide bond
conformation. We first focused on fluorinated Ac--Pro--NHMe analogues,
considered as tripeptide models (Figure~\ref{fig6}). We demonstrated
that the introduction of a CF$_3$ group at the C$^{\delta}$ position of
the pyrrolidine ring increased the \textit{cis} rotamer population
compared to the proline itself, the CF$_{3}$-pseudoprolines
2-($R$)-CF$_{3}{\rmPsi}$Pro (Tfm${\rmPsi}$Pro) and
2-($S$)-CF$_{3}{\rmPsi}$Pro (tfm${\rmPsi}$Pro)~\cite{55} exhibiting a
higher \textit{cis} conformer content than the 5-($R$)-CF$_{3}$-proline
(Tfm-Pro)~\cite{26}. We also showed that our CF$_{3}$-proline analogues
with ($R$)-configuration  at the C$^{\delta}$ position significantly
decreased the \textit{trans} to \textit{cis} amide bond isomerization
energy (ca. 3--5~kcal${\cdot}$mol$^{-1}$ compared to proline).

\begin{figure*}
\includegraphics{fig06}
\vspace*{4pt}
\caption{\label{fig6}\textit{Cis}--\textit{trans} ratio and rotational
energy barrier of fluorinated analogues of Ac--Pro--NHMe peptide
models.}
\vspace*{4pt}
\end{figure*}

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}(A)~Solvent effect on Ac--CF$_{3}\rmPsi$Pro--NHMe
peptide conformation. (B)~Main electrostatic interactions and
hyperconjugations observed in Ac--tfm$\rmPsi$Pro--NHMe.
(C)~Electrostatic contacts in the transition states of
Ac--tfm$\rmPsi$Pro--NHMe and Ac--Tfm$\rmPsi$Pro--NHMe. Reprinted with
permission from~\cite{55}. Copyright 2012 American Chemical
Society.}
\vspace*{5pt}
\end{figure*}

In collaboration with Prof.~Y.~K.~Kang and Dr.~E.~Miclet, we conducted
NMR studies and theoretical calculations on our model peptides
Ac--CF$_{3}\rmPsi$Pro--NHMe in chloroform, DMSO and water,
allowing for characterizing the solvent effect on their conformational
properties~\cite{55}. In particular, we showed the ability of the
CF$_3$ group to control the ${\uppsi}$-dihedral angle. In CHCl$_{3}$,
the 2-($R$)-CF$_{3}\rmPsi$Pro (Tfm${\rmPsi}$Pro) significantly
stabilized a ${\upgamma}$}-turn--like conformation, whereas
2-($S$)-CF$_{3}{\rmPsi}$Pro (tfm${\rmPsi}$Pro) stabilized polyproline
I (PPI)/polyproline II (PPII)-like backbone conformations
(Figure~\ref{fig7}A). These same PPI/PPII conformations were highly
dominating in water (${>}$90\%), whatever the CF$_{3}{\rmPsi}$Pro
configuration. The proline effect is therefore greatly enhanced using
trifluoromethylated pseudoprolines since the PPI/PPII-like population
in water is only ${\sim}$50\% for the natural prolyl residue.
We also observed that the C$^{\delta}$--CF$_3$
configuration could be used to efficiently constrain the ring puckering
without affecting the \textit{cis/trans} population ratio. The
2-($R$)-CF$_{3}{\rmPsi}$Pro (Tfm${\rmPsi}$Pro) and the
2-($S$)-CF$_{3}{\rmPsi}$Pro (tfm${\rmPsi}$Pro) exhibit a strong
preference for the O$^{\gamma}$-exo (up) and the O$^{\gamma}$}{-endo
(down) puckering, respectively}{. Minimized structures have proven that
both electrostatic interactions and hyperconjugations involving the
CF$_3$ group were responsible for these unique structural properties
(Figure~\ref{fig7}B)~\cite{55}. Analysis of the transition states have
shown that the favorable F}${\cdots}$HN electrostatic contact observed
in Ac--Tfm${\rmPsi}$Pro--NHMe was replaced by an unfavorable
$\mathrm{H}^{\alpha}{{\cdots}}\mathrm{HN}$ contact in
Ac--tfm${\rmPsi}$Pro--NHMe, providing a rationale for the low
\textit{cis}--\textit{trans} energy barrier observed in
Ac--Tfm${\rmPsi}$Pro--NHMe (Figure~\ref{fig7}C).

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig08}
\vspace*{-6pt}
\caption{\label{fig8}(A)~NMR parameters and conformational features of
Ac--dfm${\rmPsi}$Pro--NHMe. (B) Conformational features of
Ac--dfm${\rmPsi}$Pro--NHMe and Ac--Dfm${\rmPsi}$Pro--NHMe.}
\vspace*{6pt}
\end{figure*}


Replacing the CF$_3$ group by the CF$_{2}$H on pseudoprolines
promoted an amide bond conformation in favour of the \textit{cis}
rotamer in D$_{2}$O regardless the CF$_{2}$H${\rmPsi}$Pro
configuration (Figure~\ref{fig8})~\cite{29}. This conformational feature
was understood by analyzing the unusual NMR parameters of the
CF$_{2}$H groups (Figure~\ref{fig8}A). The \textit{cis} conformers of
the 2-($R$)-CF$_{2}$H${\rmPsi}$Pro (Dfm${\rmPsi}$Pro) and
2-($S$)-CF$_{2}$H${\rmPsi}$Pro (dfm${\rmPsi}$Pro) displayed high
chemical shift difference ($\rmDelta\delta$) of the
diastereotopic $^{19}$F as well as typical $^1$H vicinal couplings,
both reflecting a very limited rotameric averaging of the CF$_{2}$H
group. Such a restricted rotation of the CF$_{2}$H group likely arose
from an intramolecular H-bonding, the oxygen of the preceding acetyl
group facing the positively charged CF$_{2}$H proton in the
\textit{cis} conformation. Given that the \textit{cis} conformer
remains the majority in D$_{2}$O, we suggested that such an
intramolecular H-bond is endowed with a hydrophobic character. While
the 2-($S$)-CF$_{2}$H$\rmPsi$Pro (dfm$\rmPsi$Pro) displays
the same preference for the O$^{\gamma}$-endo (down) puckering as
observed with its CF$_3$ analogue, the
2-($R$)-CF$_{2}${H}$\rmPsi$Pro (Dfm$\rmPsi$Pro) shows
different puckering preferences between the \textit{cis} and the
\textit{trans} conformers (Figure~\ref{fig8}B)~\cite{29}. This
puckering switch seems to be required to allow the intramolecular
$\mathrm{NH}{{\cdots}}\mathrm{O}{=}\mathrm{C}$ H-bond in the \textit{trans}
form.

\begin{figure*}
\includegraphics{fig09}
\vspace*{-6pt}
\caption{\label{fig9}\textit{Cis/trans} populations and
puckering conformation of Fmoc-Xaa--Tfm${\rmPsi}$Pro-OMe.}
\vspace*{-6pt}
\end{figure*}

\begin{figure*}
\vspace*{2pt}
\includegraphics{fig10}
\caption{\label{fig10}(A)~Puckering preferences of Pro, Hyp,
tfm${\rmPsi}$Pro and Tfm${\rmPsi}$Pro residues. (B)
\textit{Cis}--\textit{trans} populations (\%) and puckering
preferences for collagen triplet model peptides.}
\vspace*{2pt}
\end{figure*}


Later, we reported the conformational study realized on several
heterochiral or homochiral Fmoc-Xaa--Tfm${\rmPsi}$Pro-OMe dipeptides,
where Xaa ${=}$ Gly, Ala, Val, Pro, or Aib~\cite{44}. The results
revealed an interdependence of the stereochemistry of the amino acid
preceding the CF$_{3}{\rmPsi}$Pro residue, the ring puckering and the
\textit{cis}--\textit{trans} ratio of the prolyl bond
(Figure~\ref{fig9}). While homochiral dipeptides exhibit strong
preference for the \textit{trans} amide bond, heterochiral analogues
favoured the \textit{cis} amide bond. Interestingly, homo- and
heterochiral dipeptides exhibit marked imbalance in conformer
populations with opposite \textit{cis}/\textit{trans} ratio which is
closely related with the steric hindrance of the amino acid side chain.
The pyrrolidine puckering of each conformer was then \mbox{investigated} by
NMR spectroscopy using the measurement of coupling 
constants $^{3}J_{\mathrm{H}\upbeta\ndash\mathrm{H}\upalpha}$. It appeared
that the puckering was strongly correlated with the prolyl amide bond
since \textit{trans} amide bond favored a O$^{\gamma}$-exo puckering
and \textit{cis} amide bond stabilized a O$^{\gamma}$-endo puckering.


\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig11}
\vspace*{-5pt}
\caption{\label{fig11}(A) Chemical structure of fluorinated host--guest
CMPs. (B)~Conformation of the homo-trimer helix
of~[(Pro--Hyp--Gly)$_{3}$--tfm${\rmPsi}$Pro--Tfm${\rmPsi}$Pro--Gly--(Pro--Hyp--Gly)$_{3}$] 
after a 500 ns simulation in explicit solvent. The initial structure
was derived from the 1K6F PDB structure. (C)~Circular dichroism
spectroscopy of fluorinated CMP
[(Pro--Hyp--Gly)$_{3}$--Pro--Tfm${\rmPsi}$Pro--Gly--(Pro--Hyp--Gly)$_{3}$] 
(blue squares) and
[(Pro--Hyp--Gly)$_{3}$--tfm${\rmPsi}$Pro--Tfm${\rmPsi}$Pro--Gly--(Pro--Hyp--Gly)$_{3}$]
(red circles). (a) CD spectra recorded at 4~\textdegree C. (b) Thermal
denaturation of CMPs followed by the molar ellipticity at 225 nm.
(B) and (C) are adapted from~\cite{56}. Reprinted with
permission from~\cite{56}. Copyright 2023 American Chemical Society.}
\vspace*{-5pt}
\end{figure*}

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig12}
\vspace*{-3pt}
\caption{\label{fig12}(A) Chemical structure of pseudotetrapeptide
Ac--Ala--Tfm${\rmPsi}$Pro--NHMe and overlay of the 5-lowest energy
structures obtained by molecular dynamics under NMR restraints. 
(B)~Ac--\textsc{d}-Pro--\textsc{l}-Pro--NHMe as a model for
${\upbeta}$-turn formation and set of CF$_{3}$-prolines surrogates. 
(C)~Chemical structure of structure of the fluorinated cyclic mimetic of
the CDR3 loop of Nb80.} 
\vspace*{-2pt}
\end{figure*}


We then decided to demonstrate that our fluorinated pseudoprolines can
serve as an efficient tool to control and increase the structural
stability of biomolecules and as a first example, we focused on
collagen, the most abundant protein in the animal kingdom. Because
collagen can be engineered to serve as a valuable material for
biomedical applications, numerous collagen mimetic peptides (CMPs) have
been engineered. They are based on the {repetition} of the triplet
Pro--Hyp--Gly, used to mimic the primary sequence of collagen, and
\mbox{contain} proline derivatives substituted at their C$^{\gamma}$ and/or
C$^{\beta}$  position to stabilize or functionalize \mbox{collagen}
triple-helix mimics. Using CD, NMR, and molecular dynamics (MD)
studies, we first demonstrated on collagen triplet models that the
2-($R$)-CF$_{3}{\rmPsi}$Pro (Tfm${\rmPsi}$Pro) and the
2-($S$)-CF$_{3}{\rmPsi}$Pro (tfm${\rmPsi}$Pro), when properly arranged,
meet the structural requirements for a triple-helix assembly
(Figure~\ref{fig10}A)~\cite{56}. Despite small distortions of the
canonical ${\phi}$ and ${\Psi}$ dihedral angles, the incorporation of
the two adjacent tfm${\rmPsi}$Pro and Tfm${\rmPsi}$Pro residues
maintained a high population of the all-\textit{trans} conformation
(\textit{tt}), which is required for a triple-helix assembly
(Figure~\ref{fig10}B). Analysis of ring puckers for the \textit{tt}
conformers showed that tfm${\rmPsi}$Pro and Tfm${\rmPsi}$Pro residues
displayed the strongest propensities to stabilize the endo and exo
conformations, respectively.


MD calculations showed that incorporation of the tfm${\rmPsi}$Pro and
Tfm${\rmPsi}$Pro in a host--guest CMP sequence slightly affects the
hydrogen-bond network in the close neighborhood of the substitutions.
Nevertheless, the triple-helix model remained stable with all six
trifluoromethyl groups pointing outward from the triple helix
(Figure~\ref{fig11}A). Therefore, we decided to synthesize two
host--guest CMPs (Figure~\ref{fig11}B). Both CD and $^{19}$F NMR
spectroscopy were used to demonstrate the existence of the fluorinated
triple helix in solution, which, according to the literature, was,
however, less stable than its parent CMP (Figure~\ref{fig11}C). These
results demonstrated for the first time that C$^{\delta}$-substituted
proline analogues can be incorporated in a CMP sequence, even for
relatively large groups as trifluoromethyl groups~\cite{56}.


Very recently trifluoromethyl pseudoprolines have been incorporated
into polyproline backbones and the result is that these incorporations
do not affect the PPII helical conformations of the peptides. The
trifluoromethyl groups provides an increased hydrophobicity of the
peptides and can act as a $^{19}$F NMR probes to demonstrate the
enzymatic stability of the PPII helix~\cite{54}. 


\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig13}
{\vspace*{3pt}}
\caption{\label{fig13}(A) Chemical structure of Harzianin HK-VI and
representative CD spectra of selectively $^{19}$F-labeled HZ analogues
in the presence of lysoMPC micelles.  (B) Chemical structure of
Aib-foldamers and solid-state structure of
Cbz--(Aib)$_{4}$--(${R}$)-TfmAla-OtBu (M helix).  Dashed boxes indicate
the positions of the native Aib that have been replaced by (${R}$)- or
(${S}$)-Tfm-Ala residue.}
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
\includegraphics{fig14}
\caption{\label{fig14}($R$)-${\upalpha}$-trifluoromethylalanine for
3-FABS monitoring of trypsin inhibition. Reprinted with permission of
Wiley-VCH from~\cite{52}, Copyright 2022.}
\end{figure*}

Proline is often found as a turn inducer in peptides or proteins. The
ability of the CF$_{3}$-pseudoprolines to stabilize ${\upbeta}$-turn
was first investigated on the tetrapeptide model
Ac--Ala--Tfm${\rmPsi}$Pro--NHMe. MD calculations and extensive NMR
analyses have been carried out in solution and revealed the presence of
a stable type-VI ${\upbeta}$-turn which is characterized by a
\textit{cis} peptide bond conformation
(Figure~\ref{fig12}A)~\cite{57}. Later, we were interested in
designing a cyclic peptide mimicking the ${\upbeta}$-hairpin structure
of the CDR3 loop of Nb80. In this context, we investigated 
\unskip\break{different}
fluorinated proline analogues of the well-established
\textsc{d}-Pro--\textsc{l}-Pro segment (pP) segment as a
${\upbeta}$-turn promoter~\cite{58}. An in silico conformational study
was first performed on a set of 10 fluorinated variants of the pP
sequence incorporating the ($S$)- or
($R$)-$\upalpha$-trifluoromethylprolines (tfmPro and TfmPro,
respectively), the ($S$)- or
($R$)-$\upalpha$-trifluoromethyl-oxazolidines (tfmOxa and TfmOxa,
respectively), or the 2-($S$)- or
2-($R$)-trifluoromethyl-pseudoprolines (tfm$\rmPsi$Pro and
Tfm${\rmPsi}$Pro, \mbox{respectively}) (Figure~\ref{fig12}B). 
Each fluorinated
variant of the \textsc{d}-Pro--Pro segment was based on the
Ac--XX$^{1}$--XX$^{2}$--NHMe pseudotetrapeptide structure, providing
therefore the \mbox{required} H-bond donor and acceptor to promote a type 
$\mathrm{II}'~{\upbeta}$-turn. Out of the investigated combinations, only
tfmPro--\textsc{l}-Pro and \textsc{d}-Pro--TfmOxa \mbox{exhibited} a strongly
stabilized ${\upbeta}$-turn conformation relative to the parent pP
segment. By grafting the \textsc{d}-Pro--TfmOxa sequence to the CDR3
loop of the nanobody Nb80, the corresponding cyclic peptide showed only
one conformer, allowing unambiguous assignment of the NMR signals. This
result appeared as an \mbox{improvement} knowing that the
\textsc{d}-Pro--\textsc{l}-Pro bearing cyclic peptidomimetic presented
a large conformational heterogeneity and multiple overlapping
resonances between major and minor conformers 
 (Figure~\ref{fig12}C).
\vspace*{-4pt}

\subsubsection{Acyclic ${\upalpha}$-Fluoroalkyl-$\upalpha$-amino acids}
\vspace*{-2pt}

C$^{\alpha}$-tetrasubstituted ${\upalpha}$-amino acids, such as
${\upalpha}$-aminoisobutyric acid (Aib), are conformationally
constrained nonproteinogenic ${\upalpha}$-amino acids. They are
commonly used in peptides as strong inducers of helical conformations,
such as ${\upalpha}$-helix or 3$_{10}$-helix. We demonstrated that the
($R$)-and ($S$)-${\upalpha}$-trifluoromethylalanine (Tfm-Ala), two
chiral fluorinated analogues of Aib, can serve as efficient
$^{19}$F-labels to elucidate by solid-state NMR spectroscopy the
backbone conformation and the helix alignment in lipid bilayers of the
harzianin HK-VI (HZ), a member of the antimicrobial peptaibol family
(Figure~\ref{fig13}A). Circular dichroism (CD) \mbox{spectroscopy} 
\mbox{confirmed}
that the secondary structure of the fluorinated analogues of harzianin
HK-VI wild type remained unperturbed~\cite{45}. Chiral
C${\upalpha}$-tetrasubstituted ${\upalpha}$-amino acids also proved to
be useful tools to control the handedness of helical structures, in
particular when incorporating into achiral Aib oligomers. We reported
the synthesis of short Aib-based foldamers bearing the ($R$)-Tfm-Ala or
($S$)-Tfm-Ala at different positions into the peptide
chain~\cite{59,60}. NMR studies, CD spectroscopy and X-ray
crystallography confirmed the stabilization of the $3^{10}$ helical
structure either in solution or in the solid-state. We demonstrated
that the chiral fluorinated Tfm-Ala residue can be employed not only as
chiral controllers to induce the screw-sense preference of short
Aib-based oligomers, but also as $^{19}$F NMR reporter to quantify the
magnitude of the helical excess (Figure~\ref{fig13}B). Interestingly,
the selectivity of the screw-sense was found to be reversed compared to
that induced by the non-fluorinated quaternary \textsc{l}-amino acid
chiral inducers due to the electronic properties of the CF$_3$ group
that favors a dipole alignment of the CF$_3$ with the helix\break
macrodipole~\cite{60}.
          
\subsection{$\alpha$-Fluoroalkyl-$\alpha$-amino acids as
$^{19}$F NMR probes} 

Orthogonal $^{19}$F-labeling for solid-state NMR reveals the
conformation and orientation of short \mbox{peptaibols} in
membranes~\cite{45}. Fluorinated peptides constitute valuable probes
for investigating by liquid or solid-state $^{19}$F NMR spectroscopy
the interactions of biological molecules since biological media are
free of fluorine atoms avoiding background signal and because $^{19}$F
isotropic chemical shift is very sensitive to small structural
perturbations. In 2022 our group showed that a small peptide
incorporating the non-natural ${\upalpha}$-quaternarized
($R$)-${\upalpha}$-trifluoromethylalanine (($R$)-${\upalpha}$-Tfm-Ala)
provides a convenient and accurate monitoring of trypsin
activity~\cite{52}. The accuracy of the 3-FABS (3-Fluorine atoms for
biochemical Screening) technique was demonstrated by the rapid
inhibition potency (IC$_{50}$) evaluation of a known trypsin inhibitor\break
(Figure~\ref{fig14}).

\section{Conclusion}\label{sec4}

In summary, we have developed over the past 20 years several highly
efficient synthetic methods for the synthesis of enantiopure chiral
fluorinated compounds mainly in the amino acids series and their
derivatives. The synthesis of these compounds is still a highly
challenging task and there is a great need of new robust and highly
stereoselective methods for their preparation. We demonstrated that
these compounds provided original advances in asymmetric synthesis and
peptide chemistry and biology. It is anticipated that further
development could display highly promising opportunities in
organocatalysis, medicinal chemistry and chemical biology.

\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*{Acknowledgements}

The authors warmly thank all the students, PhD students, post-docs and
colleagues who contributed to these works. The French Ministry of
Education and Research, CY Cergy Paris Universit\'{e}, the CNRS, the
ANR, the CY Initiative of Excellence (grant ``Investissements
d'Avenir'') and the French Fluorine Network (GIS-FLUOR) are thanked for
their financial support. 

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