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\DOI{10.5802/crchim.246}
\datereceived{2023-04-04}
\daterevised{2023-07-03}
\dateaccepted{2023-07-19}
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\dateposted{2023-10-19}
\begin{document}

\begin{noXML}

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

\title{Synthesis of trehalose-based chemical tools for the study of the
mycobacterial membrane}

\alttitle{Synth\`ese d'outils chimiques \`a base de tr\'ehalose pour l'\'etude
de la membrane des mycobact\'eries}

\author{\firstname{Emilie} \lastname{Lesur}\CDRorcid{0009-0000-6729-1701}}
\address{Universit\'{e} Paris-Saclay, CNRS, Institut de Chimie
Mol\'{e}culaire et des Mat\'{e}riaux d'Orsay, UMR 8182, 91405, Orsay,
France}
\email[E. Lesur]{emilie.lesur@free.fr}

\author{\firstname{Paulin} \lastname{Rollando}}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CNRS, Institut de Chimie
Mol\'{e}culaire et des Mat\'{e}riaux d'Orsay, UMR 8182, 91405, Orsay, France}
\email[P. Rollando]{paulin.rollando@universite-paris-saclay.fr}

\author{\firstname{Dominique} \lastname{Guianvarc'h}\CDRorcid{0000-0002-7722-370X}}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CNRS, Institut de Chimie
Mol\'{e}culaire et des Mat\'{e}riaux d'Orsay, UMR 8182, 91405, Orsay,
France}
\email[D. Guianvarc'h]{dominique.guianvarch@universite-paris-saclay.fr}

\author{\firstname{Yann} \lastname{Bourdreux}\CDRorcid{0000-0001-6653-7685}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CNRS, Institut de Chimie
Mol\'{e}culaire et des Mat\'{e}riaux d'Orsay, UMR 8182, 91405, Orsay,
France}
\email[Y. Bourdreux]{yann.bourdreux@universite-paris-saclay.fr}

\thanks{Agence Nationale de la Recherche (PTMyco, grant NO. ANR-22-CE44-0005-03)}

\CDRGrant[ANR]{ANR-22-CE44-0005-03}

\shortrunauthors

\begin{abstract}
Corynebacteriales including the causative agent of many diseases such
as tuberculosis are known to be extremely resistant against external
stress as well as to antibiotic treatments which is believed to be
related to the singular architecture of their mycomembrane. Over the
last decades, both bioorthogonal chemical reporters and fluorescent
probes for the metabolic labeling of bacterial cell glycans were
developed including several trehalose-based probes to study the
dynamics of mycomembrane components. This review presents an exhaustive
view on the reported syntheses of trehalose-based probes enabling the
study of the mycomembrane biogenesis.
\end{abstract}

\begin{altabstract}
Les Corynebacteriales sont des bact\'eries, dont certaines sont
responsables de maladies infectieuses telle que la tuberculose, connues
pour \^etre extr\^emement r\'esistantes aux stress externes et aux
traitements antibiotiques. Cette r\'esistance est due \`a l'architecture
particuli\`ere de leur membrane externe, la mycomembrane. Depuis
plusieurs ann\'ees des sondes fluorescentes et des rapporteurs chimiques
ont \'et\'e d\'evelopp\'es pour la marquage m\'etabolique de biomol\'ecules dans
leur environnement cellulaire. Ces outils incluent des d\'eriv\'es \`a base
de tr\'ehalose pour l'\'etude de la mycomembrane. Cette revue pr\'esente les
synt\`eses d'outils chimiques \`a base de tr\'ehalose d\'ecrits pour \'etudier
la biogen\`ese de la mycomembrane.
\end{altabstract}

\keywords{\kwd{Chemical reporter}
\kwd{Metabolic labeling}
\kwd{Bioorthogonal probe}
\kwd{Trehalose}
\kwd{Mycomembrane}
\kwd{Corynebacteriales}}

\altkeywords{\kwd{Rapporteur chimique}
\kwd{Marquage m\'etabolique}
\kwd{Sonde bioorthogonale}
\kwd{Tr\'ehalose}
\kwd{Mycomembrane}
\kwd{Corynebacteriales}}

\maketitle

\vspace{-1em}
\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

{Corynebacteriales} belong to \textit{Actinobacteria} and
include numerus bacteria responsible of human diseases such as leprosy
(\textit{Mycobacterium leprae}), diphtheria (\textit{Corynebacterium
diphtheriae}) and tuberculosis (\textit{Mycobacterium tuberculosis}).
Nowadays, the latter is still responsible for more than 10 million
infections per year~\cite{1}. In contrast, some Corynebacteriales are
non-pathogenic, such as \textit{Corynebacterium glutamicum} which is
involved in the industrial production of glutamate~\cite{2}. All
bacteria of this group possess an atypical cell envelope exceptionally
resistant against external stress and antibiotics, this resistance
being attributed to the unique organization of their cell envelope. In
addition to the plasma membrane, this envelope is composed of a
cross-linked peptidoglycan layer (PG) bounded to an arabinogalactan
(AG) complex itself connected to long fatty acids, called mycolic acids
(MA) through an ester linkage. The esterified arabinogalatan
constitutes the inner part of the outer membrane, the so called
mycomembrane (Figure~\ref{fig1}). 

\begin{figure*}
\vspace*{-1pt}
\includegraphics{fig01}
\vspace*{-1pt}
\caption{\label{fig1}Schematic representation of the cell envelope of
Corynebacteriales. (MA: mycolic acid; TDM: trehalose dimycolate; TMM:
trehalose monomycolate.)}
\vspace*{-1pt}
\end{figure*}

Mycolic acids (MA) are very long chain (C30--C90) ${\upalpha}$-branched
and ${\upbeta}$-hydroxylated fatty acids, with an
\textit{anti}-relationship between the ${\upbeta}$-OH and the
${\upalpha}$-ramification. Beyond the mycoloylated AG (AGM), the outer
leaflet of the mycomembrane is composed of non-covalently attached
glycolipids esterified with mycolic acids. These glycolipids are mainly
trehalose monomycolate (TMM) and trehalose{\unskip\break} dimycolate (TDM). After
being biosynthesized in the cytoplasm, mycolic acids are esterified to
trehalose by the polyketide synthase Pks13, giving rise to TMM, and
then transferred across the inner membrane by a transporter of the
MmpL3 family. TMM is then processed by enzymes called
mycoloyltransferases, and the mycolate part of TMM is transferred to
different acceptors. Four mycoloyltransferases have been identified in
\textit{M. tuberculosis} (Ag85), six in \textit{M. smegmatis} (Fbp) and
six in \textit{C. glutamicum} (Myt)~\cite{3}. TMM plays the role of a
mycolate donor for several mycolate acceptors and the mycolate part can
be transferred to (i) another TMM to form trehalose dimycolate (TDM);
(ii) arabinogalactan; and (iii) channel-forming porins in some
cases~\cite{4,4a,4b} (Figure~\ref{fig2}).\looseness=-1

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Schematic representation of the mycoloylation of
various acceptors in the mycomembrane and labeling localization
depending on the pathways used (green or red stars). (AG:
arabinogalactan; AGM: mycoloylated arabinogalactan; Ag85: antigen 85;
CmpL: corynebacterial membrane proteins large; MmpL: mycobacterial
membrane proteins large; Myt: mycoloyltransferase; Pks: polyketide
synthase; TMM: trehalose monomycolate; Tre: trehalose.)\vspace*{-2pt}}
\end{figure*}

Due to its importance for mycobacteria, the mycomembrane biogenesis is
highly studied. Over the last decades, several chemical reporters have
been developed to investigate biological processes in
bacteria~\cite{5,6} and in this context, mycoloyltransferases have been
widely targeted by trehalose-~and TMM-based chemical reporters.
Chemical labeling of the mycomembrane involves incorporation of
chemical tools in the cell envelope and processing of these metabolic
analogues by enzymes. The bacteria labeling can be performed in one or
two steps, depending on the analogues used. It can be a detectable
analogue of a mycomembrane metabolite, for example a mycomembrane
precursor analogue conjugated with a fluorescent moiety. In such
approach, the labeling is performed in one step allowing a direct
mycomembrane imaging, but precautions should be taken in the design of
such compounds since a bulky fluorophore could disrupt enzymatic
processes. In another approach, the metabolic precursor analogues can
be conjugated to a small bioorthogonal group, allowing the introduction
of a detectable moiety in a distinct step using a bioorthogonal
reaction with a fluorescent probe. Since the size of the bioorthogonal
groups is small compared to fluorophores, this approach, called the
bioorthogonal chemical reporter strategy, is supposed to be less
disruptive for enzymes. From a metabolic point of view, the synthetic
trehalose-based derivatives (Figure~\ref{fig2}, dashed gray box at
top) can be incorporated through the trehalose transporter LpqY-SugABC
and then coupled to a mycolic acid and transferred into the
mycomembrane, allowing specific labeling of the upper part of the
mycomembrane, i.e., the TDM layer (Figure~\ref{fig2}, red
star). Alternatively, the TMM-based derivatives (Figure~\ref{fig2},
dashed gray box at bottom), can be either directly inserted into the
mycomembrane or processed by mycoloyltransferases, thus labeling both
the inner and the outer part of the mycomembrane, AGM and TDM layers,
respectively (Figure~\ref{fig2}, green stars).\looseness=-1

Trehalose is a non-reducing and $C_{2}$ symmetrical disaccharide
composed of two glucose units with an 1,1,$\upalpha,\upalpha$-linkage.
It can be found in fungi, algae, insects, bacteria, and some
invertebrates but not in mammalian. Trehalose has been extensively
studied for its properties against stresses such as, for example, heat,
oxidative stresses, or  desiccation~\cite{7,7a}. Due to its symmetry, and
taking into account that many trehalose-containing glycoconjugates are
unsymmetrical, the most important challenges in the synthesis of
trehalose derivatives are its desymmetrization and its selective
modification among its eight hydroxyl groups. Such transformations are
clearly exposed in recent reviews and most of the syntheses rely on two
different  approaches~\cite{8,8a,8b,8c}. The first approach relies on the
formation of the trehalose core by an $\upalpha,\upalpha$-selective
glycosylation, using selectively protected glucose derivatives. The
second approach is based on the use of native trehalose as starting
material and the selective post-modification of the eight hydroxyl
groups. Both approaches are very challenging and were used for the
selective{\unskip\break}  synthesis of trehalose-based probes. In this review we will
focus on the synthesis of trehalose- and \mbox{TMM-based} probes already used
in bacteria for the study of the mycomembrane. We classified the probes
in two categories: (i) the trehalose-based probes in which the
detectable moiety or the bioorthogonal small functional group is
directly linked to the trehalose core; and (ii) the TMM-based probes and
analogues in which the detectable moiety or the bioorthogonal small
functional group is attached at the end of the lipidic chain thus
mimicking TMM. All the synthesized trehalose- and TMM-based probes,
that have been successfully used so far for metabolic labeling
experiments with diverse application, are presented in 
Figures~\ref{fig3} and~\ref{fig4} respectively.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Structures of trehalose-based probes. (Az: azide;
CDG: cephalosporinase-dependent green; Cz: carbazole; DMN:
4-($N$,$N$-Dimethylamino)-1,8-naphthalimide; 2-FD:
2-deoxy-2-fluoro; FI: fluorescein; FITC: fluorescein-isothiocyanate;
HC: hydroxychromone; NFC: nitrofuranyl calanolide; RMR: far-red
molecular rotor; TMR: tetramethylrhodamine; Tre: trehalose.)}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Structures of TMM-bases probes. (Alk: alkyne; Az:
azide; DBF: dibromofluorescein; FRET: fluorescence resonance energy
transfer; FITC: fluorescein-isothiocyanate; QTF:
quencher-trehalose-fluorophore; RMR: far-red molecular rotor; TCO:
\textit{trans}-cyclooctene; TDM: trehalose dimycolate; TMM:
trehalose monomycolate.)}
\end{figure*}

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc01}
\vspace*{3pt}
\caption{\label{sch1}Synthesis of \textbf{FITC-Tre}~\cite{9}.}
\vspace*{-10pt}
\end{scheme*}

\section{Synthesis of trehalose-based probes}\label{sec2}


The synthesis of trehalose-based probes can be either straightforward
or challenging, depending on the hydroxyl group of trehalose to be
functionalized (i.e., primary or secondary alcohol). The first
synthesis of a trehalose-based probe used in a cellular context was
reported by Davis, Barry and  \mbox{co-workers}
with the fluorescein-containing
trehalose \textbf{FITC-Tre}~\cite{9}, bearing an extra
methyl group at the anomeric position of one of the glucose units. One
of the challenging steps of the preparation of \textbf{FITC-Tre}
was the $\upalpha,\upalpha$-selective glycosylation. In this study the
authors used the $\upalpha$-selective approach developed by
Ikegami~\cite{10},  starting from the ketoside \textbf{1} as glycosyl
donor and the glucosamine derivative \textbf{2} \unskip\break as acceptor and
governed by the extra methyl group of compound \textbf{1}. The
glycosylation provided in one step the derivative \textbf{3} in 52\%
yield, functionalized with an equatorial amino 
group.
{Such glycosylations, in order to construct the
trehalose core, are very challenging, and other studies were reported
(see for example}~\cite{11,11a,11b}).  It
should be noted that the synthesis of amino-trehaloses starting from
native trehalose is very challenging and usually takes many synthetic
steps. Thus, even if the preparation of donor and acceptor \textbf{1}
and \textbf{2} could be time consuming, this approach {furnished in
one glycosylation step the expected derivative} \textbf{3}. After
methanolysis of the acetate groups and hydrogenolysis of the benzyl
carbamate, free derivative \textbf{4} was converted in
\textbf{FITC-Tre} in high yield by treatment with
fluorescein-isothiocyanate (FITC) (Scheme~\ref{sch1}). Interestingly,
this study revealed a strong Ag85 plasticity for trehalose processing
and FITC-Tre was the first trehalose analogue used in metabolic
labeling experiments on growing \textit{Mtb} producing fluorescent
bacteria.


In 2012, Bertozzi published a mycomembrane labeling study using four
azido-trehalose analogues referred to as \textbf{2}-, \textbf{3}-,
\textbf{4}- and {\textbf{6-TreAz}}~\cite{12}. 
The latter\footnote{6-TreAz was also prepared in the study of Davis,
Barry and coworkers, see reference~\cite{9}.} was
prepared according to a synthesis reported by  Hanessian~\cite{14}. The
synthesis started with the mono-\textit{O}-bromination of commercially
available $\upalpha$-$\upalpha$-\textsc{d}-trehalose in the presence of
$N$-bromosuccinimide (NBS) and triphenylphosphine. Due to the $C_{2}$
symmetry of trehalose, mono-$O$-bromination at C6 or C6$'$ gave a same
and unique derivative. Desymmetrized derivative \textbf{5} was obtained
after per-$O$-acetylation of the crude product, a step that facilitated
its purification. \textbf{5} was then engaged in a nucleophilic
substitution reaction in the presence of sodium azide followed by
de-$O$-acetylation to obtain {\textbf{6-TreAz}} (Scheme~\ref{sch2}).

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc02}
\vspace*{3pt}
\caption{\label{sch2}Synthesis of \textbf{6-TreAz}~\cite{12,14}.}
\vspace*{-1pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc03}
\vspace*{3pt}
\caption{\label{sch3}Synthesis of \textbf{2-TreAz}~\cite{12}.}
\vspace*{-5pt}
\end{scheme*}

The preparation of the azido-trehaloses functionalized through their
secondary alcohol is more challenging because it needs robust methods
enabling selective protection to differentiate the eight hydroxyl
groups. Furthermore, the introduced azido group should be positioned in
equatorial position. The synthesis of \textbf{2}- and
{\textbf{3-TreAz}} {started from the same desymmetrized intermediate}
\textbf{6} {obtained according to a procedure reported by Wallace and 
Minnikin~\cite{15}. From this common intermediate different
protection/deprotection steps were carried out to selectively isolate
the 2-~or 3-hydroxyl groups. In order to get \textbf{2-TreAz}, compound
\textbf{6} was first protected on its C2 position \textit{via} a
selective esterification on the more reactive OH-2, leading to key
compound \textbf{7} in good yield. The OH-3 was then protected using}
methoxymethyl chloride (MOMCl) and derivative \textbf{8} {was obtained
after de-$O$-benzoylation. This sequence resulted in the isolation of
the 2-hydroxyl group, which was engaged on a double 
inversion process
using the Lattrell--Dax nitrite-mediated \mbox{inversion~\cite{16,16a}.}  After
activation of the hydroxyl group of \textbf{8} using triflic anhydride,
compound \textbf{9} was obtained after treatment in the presence of
NaNO$_2$. This sequence allowed the authors to isolate a protected
trehalose with an axial hydroxyl on C2 position. Compound} \textbf{9}
was then reacted with triflic anhydride followed by lithium azide, and
the azido derivative \textbf{10} was obtained in 45\% yield. The
difficulty of this sequence could be the separation of the target
compound which can be obtained as a mixture with a side product
resulting from an elimination reaction. Finally, acidic hydrolysis
afforded \textbf{2-TreAz} with 69\% yield (Scheme~\ref{sch3}).



For the preparation of \textbf{3-TreAz}, Swern oxidation of key
intermediate \textbf{7} led to \textbf{11} in excellent yield, and then
derivative \textbf{12}, with an axial OH group at C3 was obtained through
the selective reduction of the ketone function of \textbf{11}. Then,
activation of the OH group of \textbf{12} using triflic anhydride
followed by nucleophilic substitution of the triflate intermediate by
lithium azide gave compound \textbf{13} in good yield. Finally,
de-\textit{O}-benzoylation and acidic hydrolysis led to expected
\textbf{3-TreAz} (Scheme~\ref{sch4}).

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc04}
\vspace*{3pt}
\caption{\label{sch4}Synthesis of \textbf{3-TreAz}~\cite{12}.}
\vspace*{-3pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc05}
\vspace*{3pt}
\caption{\label{sch5}Synthesis of \textbf{4-TreAz}~\cite{12}.}
\vspace*{-5pt}
\end{scheme*}


In the same study, the azido analogue, \textbf{4-TreAz} was prepared
using the hepta-\textit{O}-benzoate derivative \textbf{14} described by
Nashed and coworkers in 1993~\cite{17}.  Indeed, treatment of trehalose
by eight equivalents of benzoyl chloride led to the desymmetrized
trehalose \textbf{14} in good yield. It should be noted that, in these
conditions, two other symmetrical derivatives were also obtained,
i.e., the per-\textit{O}-benzoylated trehalose and the
$2,2',3,3',6,6'$-hexa-\textit{O}-benzoylated trehalose. The azide group
was then incorporated through a double inversion using the same
approach applied for \textbf{2-TreAz} with the Lattrell--Dax
nitrite-mediated{\unskip\break} inversion. Finally, \textbf{4-TreAz} was obtained
after de-\textit{O}-benzoylation (Scheme~\ref{sch5}).

All the four azido-trehalose analogues were used in metabolic labeling
experiments in \textit{M. smegmatis} strains using a two-step approach
through the bioorthogonal chemical reporter strategy. The derivatives
\textbf{2}- and \textbf{6-TreAz} were found to be the most efficient.
Bertozzi next used these analogues as key precursors for the synthesis
of new fluorescent trehaloses. Indeed, \textbf{TreAz} derivatives were
subjected to hydrogenolysis to give the corresponding amino-trehaloses.
All the four obtained amino-trehaloses were then reacted with
fluorescein isothiocyanate (FITC) to afford \textbf{2}-, \textbf{3}-,
\textbf{4}- and \textbf{6-FITre} (Scheme~\ref{sch6})~\cite{18}. These
fluorescein-modified trehalose probes were more efficient in metabolic
labeling experiments on Mycobacteria compared to the first reported
\textbf{FITC-Tre}. The same approach was performed to obtain the
tetramethylrhodamine-trehalose \textbf{6-TMR-Tre} for super-resolution
microscopy experiments~\cite{19}. Finally, in order to avoid the wash
steps usually performed when using fluorescent probes, the
solvatochromic dye \textbf{DMN-Tre}~\cite{20}  was prepared starting
from 6-amino-trehalose (Scheme~\ref{sch6}). Indeed, solvatochromic
tools show high shifts of fluorescence with solvent polarity
modifications and are thus efficient for mycomembrane imaging without
washing steps.

In 2014, the Swarts's group published a new route for the synthesis of
azido trehalose derivatives using a chemoenzymatic approach starting
from several azido-glucoses and uridine diphosphate-glucose
(UDP-glucose) in presence of trehalose synthase TreT
(Scheme~\ref{sch7})~\cite{21}. Under these conditions, access to
\textbf{3}- and \textbf{6-TreAz} were improved in comparison to
the firstly reported chemical syntheses. Unfortunately, this enzymatic
approach failed for the 2- and 4-modified trehaloses. For the latter,
the chemical synthesis remains still more efficient. Additionally, the
same team also developed an access to the  radioprobe
$^{\mathbf{18}}$\textbf{F-2-FDTre} using the same  methodology~\cite{22}.
\looseness=1

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc06}
\vspace*{5pt}
\caption{\label{sch6}Syntheses of \textbf{2}-, \textbf{3}-,
\textbf{4}-, \textbf{6-FITre}~\cite{18};  \textbf{6-TMR-Tre}~\cite{19}
and \textbf{DMN-Tre}~\cite{20}.  (TAMRA-NHS:
5-Carboxy-tetramethylrhodamine \textit{N}-succinimidyl ester.)}
\end{scheme*}

\begin{scheme*}
\vspace*{3pt}
\includegraphics{sc07}
\caption{\label{sch7}Chemoenzymatic approaches for the synthesis of
\textbf{3}- and \textbf{6-TreAz}~\cite{21} and
$^{\mathbf{18}}$\textbf{F-2-FDTre}~\cite{22}.}
\vspace*{-3pt}
\end{scheme*}

In 2020, the Fulham group employed this enzymatic approach for the
synthesis of 6-azido-modified derivatives with the use of
6-azido-mannose\break and 6-azido-galactose in place of
6-azido-glucose \cite{23}.  This resulted in the obtention of the
non-symmetrical trehalose analogues {\textbf{6-MannoTreAz}} and
\textbf{6-GalactoTreAz} (Scheme~\ref{sch8}). These bioorthogonal
trehalose analogues were used in \textit{M. smegmatis} labeling
experiments, highlighting a good substrate tolerance of
mycoloyltransferases.


\begin{scheme}
\includegraphics{sc08}
\vspace*{8pt}
\caption{\label{sch8}Chemoenzymatic synthesis of \textbf{6-MannoTreAz}
and \textbf{6-GalactoTreAz}~\cite{23}.}
\end{scheme}

\begin{scheme*}
\includegraphics{sc09}
\vspace*{5pt}
\caption{\label{sch9}Synthesis of \textbf{CDG-Tre} probe targeting two
mycobacterial enzymes~\cite{24}.}
\vspace*{-3pt}
\end{scheme*}

Still in 2020, the \textbf{6-TreAz} derivative was used, by Rao group,
as a precursor for the preparation of the new fluorogenic probe
\textbf{CDG-Tre} activated by the $\upbeta$-lactamase BlaC expressed in
\textit{M. tuberculosis}~\cite{24}. In this approach, the fluorophore
is quenched by the cephalosporin residue and the fluorescence is
recovered after processing by the $\upbeta$-lactamase BlaC. The
resulting fluorescent trehalose can then be\unskip\break processed by
mycoloyltransferases and thus label the mycomembrane. Freshly prepared
fluorescein derivative \textbf{16} and cephalosporin scaffold
\textbf{17} were engaged in nucleophilic substitution followed by
treatment under acidic conditions, leading to \textbf{18}. The crude
was then directly coupled without \mbox{purification} to \textbf{6-TreAz}
\textit{via} copper-catalyzed azide-alkyne cycloaddition (CuAAC)
(Scheme~\ref{sch9}).

Later, Sun and Liu used \textbf{6-TreAz} as precursor for the synthesis
of the additional fluorescent trehalose-based probe
\textbf{NFC-Tre-5}~\cite{25}. This probe featured an off-on fluorescent
group activated by the nitroreductase Rv2466 allowing the specific
labeling of single cells. \textbf{NFC-Tre-5} was obtained after CuAAC
reaction between \textbf{6-TreAz} and an alkyne-modified nitrofuranyl
calanolide scaffold (Scheme~\ref{sch10}).

\begin{scheme*}
\includegraphics{sc10}
\vspace*{5pt}
\caption{\label{sch10}Synthesis of \textbf{NFC-Tre-5}~\cite{25}.}
\vspace*{-3pt}
\end{scheme*}

In 2022, Guianvarc'h, Vauzeilles and Bourdreux reported the synthesis
of two new trehalose-based probes modified at C2 for the metabolic
labeling of the mycomembrane~\cite{26} i.e., a native trehalose bearing
a short ether linker functionalized with a fluorescein,
\textbf{2-FIC5Tre}, and \textbf{2-epi-TreAz}, an epimer at C2 of
the known \textbf{2-TreAz}. In the course of this study, they also
proposed a new route for the synthesis of \textbf{2-TreAz}. The key
step of these syntheses was a tandem one-pot protection and
desymmetrization of trehalose mediated by
$\mathrm{FeCl}_{3}{\cdot}6\mathrm{H}_{2}\mathrm{O}$. In this study, the
authors revisited a first published  protocol~\cite{27} in order to get
a protected trehalose with only a free OH group at C2 position.
Briefly, $\upalpha,\upalpha$-\textsc{d}-trehalose was firstly per-$O$-silylated
in pyridine and the resulting derivative \textbf{19} was engaged, in a
tandem protection protocol mediated by
$\mathrm{FeCl}_{3}{\cdot}6\mathrm{H}_2\mathrm{O}$ in the presence of
benzaldehyde and triethylsilane (Scheme~\ref{sch11}). Under these
conditions the desymmetrized and protected trehalose \textbf{20} was
obtained in 40\% yield. This key compound was used as starting material
for the synthesis of the three derivatives \textbf{2-epi-TreAz},
\textbf{2-TreAz} and \textbf{2-FIC5Tre}.
The tri-$O$-benzylated trehalose \textbf{20} {was therefore converted
into triflate \textbf{21} and then immediately engaged in a
nucleophilic substitution reaction to introduce the azido moiety in
presence of} tetra-\textit{n}-butylammonium azide (TBAN$_{3}$). This
sequence allowed to rapidly obtain an axial azido moiety at C2 of
trehalose. Challenging deprotections were then carried out using
anhydrous iron(\textsc{iii}) chloride in the presence of acetic
anhydride followed by de-$O$-acetylation providing
\textbf{2-epi-TreAz} in good yield (Scheme~\ref{sch12}). The known
\textbf{2-TreAz} was also synthesized during this study by
performing the same double inversion  protocol as the one used for its
first synthesis by the group of Bertozzi. Triflate \textbf{21} was
indeed treated with sodium nitrite and the resulting compound
\textbf{23} with an axial OH group was then converted into
\textbf{24} after an activation-nucleophilic substitution sequence
using triflate anhydride in pyridine followed by a TBAN$_{3}$
treatment. Then, the same deprotection protocols were performed
leading to \textbf{2-TreAz}. Finally, the authors also prepared a
fluorescent probe starting from \textbf{20}. A small linker bearing
an azido moiety was first introduced by alkylation of the hydroxyl
group and the resulting compound \textbf{25} was subjected to
hydrogenolysis. Then, the amine function of \textbf{26} was coupled
to the fluorophore using a treatment with FITC leading to
\textbf{2-FIC5Tre}. The three probes were successfully used in
mycomembrane labeling experiments on \textit{C. glutamicum}.
\textbf{2-TreAz} and \textbf{2-epi-TreAz} appeared to have similar
efficacy again indicating a good substrate tolerance of
mycoloyltransferases.\looseness=1

\begin{scheme*}
\includegraphics{sc11}
\vspace*{5pt}
\caption{\label{sch11}$\mathrm{FeCl}_{3}{\cdot}6\mathrm{H}_{2}\mathrm{O}$ 
mediated one-pot desymmetrization of trehalose~\cite{26,27}.}
\vspace*{-6pt}
\end{scheme*}

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc12}
\vspace*{3pt}
\caption{\label{sch12}Syntheses of \textbf{2-epi-TreAz};
\textbf{2-TreAz} and \textbf{2-FIC5Tre}~\cite{26}.}
\vspace*{-5pt}
\end{scheme*}

Recently, Bertozzi published the synthesis of new solvatochromic
trehalose-based probes featuring 3-hydroxychromone (3HC) dyes,
\textbf{3HC-3-Tre} and \textbf{3HC-2-Tre}, the first displaying a
10-fold increase in fluorescence intensity compared to the previous
report on \textbf{DMN-Tre}~\cite{28}.
These
fluorescent probes are known to have high fluorescence quantum yields
and they proved to efficiently label \textit{M. tuberculosis} cells
within 10 min of probe treatment. For these syntheses, the authors took
advantage of the known desymmetrized and monobrominated derivative
\textbf{5} previously described by Hanessian. Compound \textbf{5} was
engaged in nucleophilic substitution with the two {\unskip\break} 3-hydroxychromones
\textbf{27} and \textbf{28}, providing mono-$O$-coupled derivatives
\textbf{29} and \textbf{30}. \textbf{3HC-3-Tre} and {\textbf{3HC-2-Tre}
were finally obtained after de-$O$-acetylation with sodium methoxide
(Scheme~\ref{sch13}).

\begin{scheme*}
\includegraphics{sc13}
\vspace*{3pt}
\caption{\label{sch13}Synthesis of \textbf{3HC-2-Tre} and
\textbf{3HC-3-Tre}~\cite{28}.}
\vspace*{-8pt}
\end{scheme*}

Very recently, Swarts's lab added a new fluorogenic trehalose analogue
to the trehalose-based probes collection~\cite{29}. This probe,
\textbf{RMR-Tre}, featured\break a molecular rotor allowing turn-on far-red
fluorescence sensor upon interaction with constraint environment such
as the mycomembrane. Derivative \textbf{31}, obtained after Knoevenagel
condensation between the known aldehyde} \textbf{32} and malonitrile,
was coupled, using
${N},{N},N^{\prime},N^{\prime}$-tetramethyl-$O$-($N$-succinimidyl)uronium 
tetrafluoroborate (TSTU), to 6-amino trehalose \textbf{33}  providing
{\textbf{RMR-Tre}} in good yield (Scheme~\ref{sch14}).

\begin{scheme*}
\includegraphics{sc14}
\vspace*{3pt}
\caption{\label{sch14}Synthesis of \textbf{RMR-Tre}~\cite{29}.}
\vspace*{-3pt}
\end{scheme*}


Finally, during the preparation of this review, Yan and coworkers
reported the synthesis of a fluorescence turn-on probe \textbf{Tre-Cz}
and its use for imaging mycobacteria after photoactivation of the aryl
azide allowing the formation of the fluorescent product \cite{30}. It
was prepared starting from the known 6-amino-trehalose \textbf{33} and
a $N$-hydroxysuccinimide(NHS)-functionalized carbazole
derivative (Scheme~\ref{sch15}).

\begin{scheme}
\vspace*{3pt}
\includegraphics{sc15}
\caption{\label{sch15}Synthesis of \textbf{Tre-Cz}~\cite{30}.\vspace*{-11pt}}
\end{scheme}


\section{Synthesis of TMM-based probes}\label{sec3}

In parallel to trehalose-based probes, other useful chemical tools,
i.e., acylated trehalose-based probes mimicking TMM, were synthesized
to label the mycomembrane. In contrast to trehalose-based probes that
are processed through the recycling trehalose pathway, TMM-based probes
target mycoloyltransferases allowing the labeling of both the outer
leaflet and the inner part of the mycomembrane (TDM and AGM layers,
respectively). 

From a synthetic point of view, different challenges have to be
addressed for the preparation of TMM-based probes. The first one is the
trehalose selective esterification of the lipidic part mimicking the
mycolate chain, which relies on the difference of reactivity between
primary and secondary alcohols. The most important challenge is the
preparation of the mycolic acid pattern which implies (i) the
control of the absolute configuration of its two stereogenic centers
and (ii) the introduction of a bioorthogonal or a detectable tag on one
of the long fatty acid chains. Some enantioselective syntheses of
mycolic acids have already been reported in the  
literature (for a few examples see \cite{31,31a,31b}), 
but the main difficulty for the preparation of such TMM-based probes is
to make them compatible with a bioorthogonal or detectable moiety.
 Several teams have been interested in the synthesis of TMM-based
chemical reporters and most of them reported simplified structures by
replacing the complex native mycolic pattern by a simple acyl chain
including the tag at the end. These probes were prepared using the same
approach for the esterification of the lipidic chain of interest with
trehalose. It relies on the per-${O}$-silylation of trehalose followed
by a controlled deprotection of the two primary positions leading to
the symmetrical diol \textbf{34} according to a procedure reported by
Toubiana~\cite{32}. Then, the deprotected derivative \textbf{34} is
monoesterified in the presence of a coupling agent and the resulting
ester is fully deprotected under acidic conditions
 (Scheme~\ref{sch16})~\cite{33}.

\begin{scheme*}
\vspace*{-3pt}
\includegraphics{sc16}
\vspace*{3pt}
\caption{\label{sch16}Approach for selective synthesis of
6-$O$-acylated trehaloses.}
\vspace*{-6pt}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc17}
\vspace*{3pt}
\caption{\label{sch17}Synthesis of {\textbf{\textit{N}}}-  and
\textbf{\textit{O}-AlkTMM}~\cite{34}.}
\vspace*{-10pt}
\end{scheme*}

In 2016, Swarts took advantage of this approach and published the first
bioorthogonal analogue related to TMM using commercially available
6-heptynoic acid as simplified mycolic acid mimic~\cite{34}. This
analogue was obtained very efficiently by reaction between compound
\textbf{34} and 6-heptynoic acid in the presence of
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The
esterified intermediate \textbf{35} was then fully deprotected under
acidic conditions leading to \textbf{\textit{O}-AlkTMM} (Scheme~\ref{sch17}).
This simplified TMM analogue was successfully used for metabolic
labeling experiments in \textit{M. smegmatis}. During this study,
6-heptynoic acid was also used for the preparation of an amide-linked
analogue. The known \textbf{6-TreAz} was first converted to compound
\textbf{33} using Staudinger reduction and the resulting amine was
coupled to 6-heptynoic acid in the presence of
${O}$-(benzotriazol-1-yl)-$N$,$N$,$N^{\prime}$,$N^{\prime}$-tetramethyluronium hexafluorophosphate 
(HBTU) affording \textbf{\textit{N}-AlkTMM}. The main difference between these
two probes is the fact that after being processed by
mycoloyltransferases, \textbf{\textit{O}-AlkTMM} can label both the inner and
the outer leaflets of the mycomembrane whereas
\textbf{\textit{N}-AlkTMM} can only label the outer part
(i.e., the TDM layer).

A few years later, the same team extended the range of TMM-based probes
by publishing a \mbox{collection} of new analogues differing by the chain
length, the bioorthogonal group or by the introduction of a
fluorophore~\cite{35}. On the basis of their previous work, the authors
prepared the two other alkyne-TMM
\textbf{\textit{O}-AlkTMM-C5} and
\textbf{\textit{O}-AlkTMM-C11} with 5 and 11 carbon atoms
on the lipidic moiety, respectively. Compound \textbf{34} was also
esterified with 10-azido-decanoic acid providing
\textbf{\textit{O}-AzTMM}, an acylated trehalose with an
azide as bioorthogonal group. This compound was finally converted to
the corresponding amine allowing the coupling to a trans-cyclooctene
\textit{N}-hydroxysuccinimide ester (NHS-TCO) and to FITC, leading
to \textbf{\textit{O}-TCO-TMM} for tetrazine ligation
and \textbf{\textit{O}-FITC-TMM} respectively
(Scheme~\ref{sch18}). Very recently, the same lab expanded the far-red
molecular rotor probe family by coupling compound \textbf{31} to
intermediate \textbf{36} furnishing \textbf{RMR-TMM}
(Scheme~\ref{sch18})~\cite{29}.


\begin{scheme*}
\includegraphics{sc18}
\vspace*{4pt}
\caption{\label{sch18}Syntheses of \textbf{\textit{O}-AzTMM}, 
\textbf{\textit{O}-TCO-TMM},
\textbf{\textit{O}-FITC-TMM}~\cite{35}  and
\textbf{RMR-TMM}~\cite{29}.}
\end{scheme*}

These studies have provided an impressive molecular diversity with
different bioorthogonal or fluorescent moieties attached to the end of
the ester-linked mono-acyl chain, but none of these included the native
pattern of mycolic acids which represents an important synthetic
challenge. In this way, Guianvarc'h and Bourdreux reported in 2019 the
first access to trehalose monomycolate bioorthogonal probes closely
mimicking the complex structure of native TMM~\cite{36}.
Both \textbf{Alk\textit{m}}$_{13}\boldsymbol
{\upalpha}_{0}$\textbf{TMM} and \textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{10}$\textbf{TMM} 
analogues featured an OH group in ${\upbeta}$ position with an
($R$) stereochemistry (see Scheme~\ref{sch20}). Moreover,
\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{10}$\textbf{TMM} 
also had the branched ${\upalpha}$-lipidic chain of mycolic acids, with
an \textit{anti}-relationship between the two ${\upalpha}$ and
${\upbeta}$ substituents. The ${\upbeta}$-ketoester \textbf{37},
obtained after a two-carbon Masamune homologation of the corresponding
alkyne-protected carboxylic acid, was engaged in an enantioselective
reduction in the presence of Noyori's catalyst
(\textit{R})-BinapRuBr$_{2}$. Under these conditions, the
triisopropylsilyl protecting group prevented the undesired concomitant
reduction of the alkyne. Thus, the expected ${\upbeta}$-hydroxyester
\textbf{38}, isolated in excellent yield and enantioselectivity, was
then alkylated in the presence of 1-iododecane and
hexamethylphosphoramide (HMPA) furnishing diastereoselectively
\textbf{39} in a moderate yield. After \textit{n}-tetrabutylammonium
fluoride (TBAF) deprotection of the alkyne, the ester was saponified
and the ${\upbeta}$-OH group was protected as triethylsilylether in
anticipation of the future esterification with trehalose, providing
carboxylic acid \textbf{42} (Scheme~\ref{sch19}). During this study, the
authors also prepared a bioorthogonal ${\upbeta}$-hydroxylated fatty
acid lacking the ${\upalpha}$-side chain of mycolic acids.
${\upbeta}$-hydroxyester \textbf{38} was firstly desilylated,
saponified and the ${\upbeta}$-OH group protected, leading to
carboxylic acid~\textbf{44}.\looseness=1

\begin{scheme*}
\includegraphics{sc19}
\vspace*{3pt}
\caption{\label{sch19}Synthesis of the first mycolic acid with an alkyne
bioorthogonal moiety~\cite{36}.}
{\vspace*{-4pt}}
\end{scheme*}

\begin{scheme*}
\vspace*{3pt}
\includegraphics{sc20}
\vspace*{3pt}
\caption{\label{sch20}Synthesis of 
\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{{0}}${\textbf{TMM}} 
and \textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{10}$\textbf{TMM}~\cite{36}.}
\end{scheme*}

\begin{scheme*}
\includegraphics{sc21}
\vspace*{3pt}
\caption{\label{sch21}Synthesis of
\textbf{\textit{O}-x-AlkTMM-C15} and
\textbf{\textit{N}-x-AlkTMM-C15}~\cite{37}.}
\end{scheme*}

Finally, the two probes
\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{10}$\textbf{TMM} 
and
\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{{0}}$\textbf{TMM} 
were obtained using the same {approach} as previously described for the
syntheses of the previous TMM analogues. Fatty acids \textbf{42} and
\textbf{44} were esterified to trehalose \textbf{34} in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and the resulting
intermediates were fully deprotected under acidic conditions, providing
the native TMM-based probes (Scheme~\ref{sch20}). These two tools
proved to be highly efficient for the labeling of the \textit{C.
glutamicum} mycomembrane even at low concentration. They are supposed
to avoid the substantial acylhydrolase activity observed with
unbranched lipid and to improve the native transfer activity of
mycolate to its acceptors.\looseness=1

In 2020, Swarts proposed the TMM-based probes,
\textbf{\textit{O}-x-AlkTMM-C15} and
\textbf{\textit{N}-x-AlkTMM-C15}, with an extra
photoactivatable diazirine into the lipidic part in order to identify
mycolate-protein interactions~\cite{37}. The synthesis of these tools
relied on the preparation of the known bifunctional fatty acid
\textbf{49}~\cite{38}. The latter was prepared by nucleophilic addition
of the Grignard \textbf{46} to acyl chloride \textbf{45} and subsequent
deprotections under alkaline conditions. The diazirine was then
installed as previously reported~\cite{39}  and coupled to trehalose
derivatives \textbf{34} and \textbf{33} providing
\textbf{\textit{O}-x-AlkTMM-C15} and
\textbf{\textit{N}-x-AlkTMM-C15}, respectively
(Scheme~\ref{sch21}).

A few years ago, Kiessling reported an ingenious strategy based on an
observed acylhydrolase activity\break of mycoloyltransferases in the presence
of TMM analogues with unbranched lipids~\cite{40}. 
\begin{scheme*}
\vspace*{-4pt}
\includegraphics{sc22}
\vspace*{2pt}
\caption{\label{sch22}Synthesis of \textbf{QTF}~\cite{41}.}
\vspace*{-8pt}
\end{scheme*}
She designed the fluorogenic \textbf{QTF}  that is structurally closer to TDM rather
than TMM analogues~\cite{41}. 
\begin{scheme*}
\includegraphics{sc23}
\vspace*{-2pt}
\caption{\label{sch23}Synthesis of \textbf{FRET-TDM}~\cite{42}.}
\vspace*{-10pt}
\end{scheme*}
Indeed, one primary alcohol of trehalose
is esterified to an acyl chain bearing a fluorescent\unskip\break 
BODIPY-based-probe, whereas the second primary alcohol is linked to a
fluorescence quencher. When~\textbf{QTF} is processed by
mycoloyltransferases, the unbranched lipid bearing the fluorophore
moiety can promote hydrolysis of the acyl-enzyme intermediate thus
restoring the fluorescence. \textbf{QTF} was prepared by two successive
selective esterifications with 4-dimethylaminoazobenzene-4-carboxylic
acid (DABCYL-OH) and 15-azidopentadecanoic acid, respectively.
Di-$O$-esterified compound \textbf{51} was next coupled to a
fluorophore through CuAAC reaction leading, after deprotection, to
\textbf{QTF} (Scheme~\ref{sch22}).



\begin{scheme*}
\vspace*{3pt}
\includegraphics{sc24}
\vspace*{3pt}
\caption{\label{sch24}Synthesis of
\textbf{\textit{O}-TMM-647},
\textbf{\textit{O}-TMM-DBF}~\cite{43}.}
\end{scheme*}

This strategy was next used by Swarts in a study targeting remodelling
enzymes in mycobacteria, such as TDM hydrolase (Tdmh)~\cite{42}. The
fluorogenic probe was obtained starting from \textbf{34} which was
first fully esterified with 10-azidodecanoic acid in the presence of
DCC and DMAP. The esterified derivative \textbf{52} was next converted
to the diamine derivative \textbf{53} by reduction of the two azido
groups. A fluorescein moiety was then introduced by treatment with FITC
and the quencher of fluorescence was installed to provide the
\textbf{FRET-TDM} probe (Scheme~\ref{sch23}).


Following its description (see Scheme~\ref{sch17}), 
\textbf{\textit{O}-AlkTMM} was used as starting material for
the preparation of the two additional fluorescent probes
\textbf{\textit{O}-TMM-647} and
\textbf{\textit{O}-TMM-DBF}{. Indeed, Theriot reported in
2019 the use of these two probes, synthesized by CuAAC reaction
between} {\textbf{\textit{O}-AlkTMM}} {and two different
azide fluorophores, in a study on} {\textit{C. glutamicum}}
(Scheme~\ref{sch24})~\cite{43}.

\begin{scheme*}
\vspace*{3pt}
\includegraphics{sc25}
\vspace*{3pt}
\caption{\label{sch25}Synthesis of 
\textbf{\textit{O}-AzAlkTMM}~\cite{44}.}
\end{scheme*}

Finally, {\textbf{6-TreAz}} and {\textbf{\textit{O}-AlkTMM}}
{were merged giving rise to the bifunctional reporter}
{\textbf{\textit{O}-AzAlkTMM}}~\cite{44}. It
was synthesized using the approach used for most of the described TMM
analogues as indicated in Scheme~\ref{sch16}, starting from
\textbf{6-TreAz}. Indeed \textbf{6-TreAz} was first per-$O$-silylated
and then treated under mild basic conditions, leading to \textbf{55}.
The free alcohol of \textbf{55} was then esterified with 6-heptynoic
acid and the resulting compound was fully deprotected to afford the
expected bifunctional chemical reporter
\textbf{\textit{O}-AzAlkTMM} (Scheme~\ref{sch25}) with an
azide moiety on the trehalose core, and an alkyne on the lipidic chain
mimicking the mycolic acid. These two bioorthogonal groups can react
successively with two different fluorophores. Furthermore, the azide
group can label only the TDM layer of the mycomembrane, whereas the
alkyne moiety can label both the TDM and AGM layers. This bifunctional
probe was used to study different metabolic processes by targeting
mycoloyltransferase activities as well as trehalose recycling process.


%tables 1 and 2
\begin{table*} %tab1
\caption{\label{tab1}Recapitulative table on the principal features and
uses of trehalose-based probes described so far}
\fontsize{8}{10}\selectfont
\tabcolsep=3pt
\begin{tabular}{lll}
\thead
Classification & \parbox[t]{3cm}{\raggedright Probe name and reference$^a$} & 
Principal features \vspace*{2pt}\\
\endthead
\morerows{3}{\raisebox{-8em}{\parbox[t]{3cm}{\raggedright\textit{Fluorescein} and tetramethylrhodamine 
\textit{conjugated trehalose}}}}  & 
\raisebox{-2.6em}{\textbf{FITC-Tre}~\cite{9}} & 
\parbox[t]{9cm}{\raggedright $\bullet$ First trehalose-based fluorescent probe
with a C1 methyl group at the anomeric position \Lbreak $\bullet$ Metabolic
labeling experiments on growing \textit{Mtb and} within infected
macrophages \Lbreak $\bullet$ Revealed a strong Ag85 plasticity}\vspace*{5pt}   \\

& \raisebox{-1.4em}{\textbf{2-,3-,4-,6-FITre}~\cite{18}} & \parbox[t]{9cm}{\raggedright $\bullet$ First natural trehalose-based
fluorescent probe \Lbreak $\bullet$ More efficient in labeling than the
FITC-Tre (\textit{Msmeg}, \textit{Cglut}) \Lbreak $\bullet$ Applied to
elucidate the mobility of labeled glycolipids in mycobacteria in
several conditions}\vspace*{5pt}  \\

& \textbf{2-FIC5Tre}~\cite{26} & \parbox[t]{9cm}{\raggedright $\bullet$ Fluorophore attached to trehalose
through an ether-linker \Lbreak $\bullet$ Enable efficient one step labeling of
mycobacteria (\textit{Cglut})} \vspace*{5pt}\\

& \raisebox{-1.4em}{\textbf{6-TMR-Tre}~\cite{19}}& \parbox[t]{9cm}{\raggedright $\bullet$ Mycomembrane visualization with
super-resolution microscopy \Lbreak $\bullet$ Study of the mycomembrane and
peptidoglycan dynamics using a dual metabolic labeling strategy
(\textit{Msmeg}, \textit{Mm}, \textit{Cglut})}\vspace*{2pt} \\\hline

\morerows{2}{\raisebox{-5em}{Azido-trehalose}}  &
\raisebox{-1.4em}{\textbf{2-,3-,4-,6-TreAz}~\cite{12,21,26}} & \parbox[t]{9cm}{\raggedright $\bullet$ First bioorthogonal
trehalose for chemical reporter strategy \Lbreak $\bullet$ Study of trehalose
metabolism in live mycobacteria (\textit{Msmeg}, \textit{Mtb}, BCG)
\Lbreak $\bullet$  2- and 6-TreAz more efficient}\vspace*{5pt} \\

& \parbox[t]{3cm}{\textbf{6-MannoTreAz}~\cite{23}\Lbreak   \textbf{6-GalactoTreAz}~\cite{23}} & 
\parbox[t]{9cm}{\raggedright $\bullet$ Good
substrate tolerance toward mycoloyltransferases \Lbreak
(\textit{Msmeg}, BCG)}\vspace*{5pt} \\

& \textbf{2-epiTreAz}~\cite{26} & \parbox[t]{9cm}{\raggedright $\bullet$ Good substrate tolerance toward
mycoloyltransferases \Lbreak $\bullet$ Similar efficiency than 2-TreAz in
labeling experiments (\textit{Cglut})}\vspace*{2pt}\\\hline

\morerows{2}{\raisebox{-8em}{\parbox[t]{3cm}{\raggedright Trehalose conjugated to solvatochromic dye}}}  &
\raisebox{-2.8em}{\textbf{DMN-Tre}~\cite{20}} & \parbox[t]{9cm}{\raggedright $\bullet$ Sensitive to changes in the polarity of
the medium  \Lbreak $\bullet$ 700-fold increase in fluorescence intensity in
hydrophobic environment \Lbreak $\bullet$ Does not require sample washing
\Lbreak $\bullet$ Strong potential to detect \textit{Mtb} in patients' sputum
\Lbreak (\textit{Mtb}, \textit{Msmeg}, \textit{Mm})}\vspace*{5pt} \\

& \parbox[t]{3cm}{\textbf{3HC-3-Tre}~\cite{28} \Lbreak \textbf{3HC-2-Tre}~\cite{28}} & 
\parbox[t]{9cm}{\raggedright $\bullet$
10-fold increase in fluorescence intensity compared to DMN-Tre \Lbreak
$\bullet$ Efficiently label \textit{Mtb} within 10 min of probe treatment
\Lbreak (\textit{Msmeg}, \textit{Mtb}, \textit{Cglut})}\vspace*{5pt} \\

& \raisebox{-2em}{\textbf{RMR-Tre}~\cite{29}} & \parbox[t]{9cm}{\raggedright $\bullet$ Rotor turn-on fluorophore, no-wash and
fast detection of live cells \Lbreak $\bullet$ Bright far-red emission and
low-background fluorescence detection  \Lbreak $\bullet$ 100-fold enhancement
in \textit{Mtb} labeling compared to previous fluorogenic trehalose
probes \Lbreak (\textit{Msmeg}, \textit{Mtb})}   \vspace*{2pt}\\\hline

\morerows{1}{\raisebox{-3.5em}{\parbox[t]{3cm}{\raggedright Trehalose conjugated to off-on fluorescent dye
activated by enzyme}}} & \raisebox{-2em}{\textbf{CDG-Tre}~\cite{24}} & \parbox[t]{9cm}{\raggedright $\bullet$ Release of the
caged fluorophore conjugated to trehalose by the ${\upbeta}$-lactamase
BlaC (\textit{Msmeg}, BCG) \Lbreak $\bullet$ low concentration for the labeling
of phagocytosed live BCG within macrophages}\vspace*{5pt} \\

& \raisebox{-2em}{\textbf{NFC-Tre-5}~\cite{25}} & \parbox[t]{9cm}{\raggedright $\bullet$ Activated by the nitroreductase
Rv2466  \Lbreak $\bullet$ Specific labeling of single cells (\textit{Mmseg},
\textit{Mtb}, BCG) \Lbreak $\bullet$ Strong potential for exploring the
relationship between the host and pathogen} \vspace*{2pt} \\\hline

\parbox[t]{3cm}{\raggedright Trehalose conjugated to off-on fluorescent dye\break activated by light} &
\raisebox{-1em}{\textbf{Tre-Cz}~\cite{30}} & \parbox[t]{9cm}{\raggedright $\bullet$ Photoactivation and detection using a
handheld UV lamp (\textit{Msmeg}, \textit{Mtb}) \Lbreak $\bullet$ Trehalose
uptake pathway hijacked: intracellular incorporation through the
LpqY-SugABC transporter} \vspace*{2pt}\\\hline

\parbox[t]{3cm}{Trehalose functionalized with radioprobe} &
$^{\mathbf{18}}$\textbf{F-2-FDTre}~\cite{22} & \parbox[t]{9cm}{\raggedright $\bullet$ Positron emission
tomography (PET) imaging to allow \textit{in vivo} imaging of trehalose
metabolism (\textit{Msmeg})}\vspace*{3pt}
\botline
\end{tabular}
\tabnote{$^a$Structures of the probes are shown in Figure~\ref{fig3}.}
\end{table*}

\begin{table*} %tab2
\caption{\label{tab2}Recapitulative table on the principal features and
uses of TMM-based probes described so far}
\fontsize{8}{9.5}\selectfont
\tabcolsep=1.7pt
\begin{tabular}{lll}
\thead
Classification & \parbox[t]{3cm}{\raggedright Probe name and
reference$^a$} & Principal features \vspace*{2pt}\\
\endthead
\morerows{1}{\raisebox{-5em}{\parbox[t]{3.5cm}{\raggedright Bioorthogonal simplified analogs of TMM}}} &
\parbox[t]{2.5cm}{\raggedright\vspace*{.5em}\textbf{\textit{O}-AlkTMM}~\cite{34} \Lbreak
\textbf{\textit{N}-AlkTMM}~\cite{34}\vspace*{4.2em}} &
\parbox[t]{9cm}{\raggedright $\bullet$ First bioorthogonal
TMM analog with unbranched acyl chain, used in metabolic labeling
experiments (\textit{Msmeg, Cglut}) \Lbreak $\bullet$ \textit{O}-AlkTMM
processed by mycoloyltransferases and enabling selective \textit{in
situ} detection of the mycomembrane components in living mycobacteria
\Lbreak $\bullet$ \textit{N}-AlkTMM suitable to analyze the outer leaflet of
the mycomembrane}  \vspace*{2pt}\\

& \parbox[t]{2.5cm}{\raggedright\textbf{\textit{O}-AlkTMM-C5}~\cite{35} \Lbreak
\textbf{\textit{O}-AlkTMM-C11}~\cite{35} \Lbreak
\textbf{\textit{O-}Az-TMM}~\cite{35} \Lbreak
\textbf{\textit{O}-TCO-TMM}~\cite{35}} &
\parbox[t]{9cm}{\raggedright $\bullet$ Expansion of the set of TMM reporter analogs
\Lbreak (\textit{Cglut}, \textit{Msmeg}) \Lbreak $\bullet$ Rapid cell labeling
(\textit{Msmeg}) through tetrazine ligation} \vspace*{2pt}\\\hline

\parbox[t]{3.5cm}{\raggedright TMM mycolic acid-based bioorthogonal reporter} &
\parbox[t]{2.5cm}{\raggedright\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{{0}}$\textbf{TMM}~\cite{36} \Lbreak 
\textbf{Alk\textit{m}}$_{13}\boldsymbol{\upalpha}_{\textbf{10}}$\textbf{TMM}~\cite{36}} 
& \parbox[t]{8.7cm}{\raggedright $\bullet$ First bioorthogonal TMM featuring the complex mycolic acid moiety
\Lbreak $\bullet$ Labeling of mycomembrane with a shorter labeling time and
very low doses (\textit{Cglut})} \vspace*{2pt}\\\hline

\morerows{2}{\raisebox{-4em}{\parbox[t]{3.5cm}{\raggedright Simplified analogs of TMM functionalized with a
fluorescent dye}}}  & \raisebox{-2em}{\textbf{\textit{O}-FITC-TMM}~\cite{35}} & 
\parbox[t]{9cm}{\raggedright  $\bullet$ First fluorescent TMM analog \Lbreak $\bullet$ Enable one step labeling of live
cells using TMM analog \Lbreak (\textit{Cglut}, \textit{Msmeg})}\vspace*{5pt} \\

& \raisebox{-2em}{\textbf{RMR-TMM}~\cite{29}} & \parbox[t]{9cm}{\raggedright $\bullet$ Far-red molecular rotor probe  \Lbreak
$\bullet$ Enable no-wash and fast detection of live cells \Lbreak
(\textit{Msmeg}, \textit{Mtb})} \vspace*{5pt}\\

& \parbox[t]{3cm}{\raggedright\textbf{\textit{O}-TMM-647}~\cite{43} \Lbreak
\textbf{\textit{O}-TMM-DBF}~\cite{43}} & \parbox[t]{9cm}{\raggedright $\bullet$ Fluorescent TMM
analog used to follow the assembly dynamics of the mycomembrane in live
cells \Lbreak (\textit{Cglut}, \textit{Msmeg})} \vspace*{2pt}\\\hline

\parbox[t]{3.5cm}{\raggedright Photoactivatable and bioorthogonal simplified analog of TMM} 
& \parbox[t]{3cm}{\raggedright \textbf{\textit{O}-x-AlkTMMC15}~\cite{37} \Lbreak
\textbf{\textit{N}-x-AlkTMMC15}~\cite{37}} & \parbox[t]{9cm}{\raggedright $\bullet$ First
photoactivatable TMM analog enabling \textit{in vivo}
photo-cross-linking and click-chemistry-mediated analysis of
mycolate-interacting proteins \Lbreak (\textit{Msmeg, Cglut})}\vspace*{2pt}\\\hline

\morerows{1}{\raisebox{-4em}{\parbox[t]{3.2cm}{\raggedright  Quencher-trehalose-{\ubreak}fluorophore probe}}} & 
\raisebox{-2em}{\textbf{QTF}~\cite{41}} &\parbox[t]{9cm}{\raggedright  $\bullet$ First FRET trehalose mycolate-based
probe  \Lbreak (\textit{Mtb}, \textit{Msmeg}, \textit{Cglut}) \Lbreak $\bullet$
Unbranched lipid bearing the fluorophore moiety to promote hydrolysis
of the acyl-enzyme intermediate enabling real-time imaging in native
cellular environment} \vspace*{8pt}\\

& \raisebox{-2em}{\textbf{FRET-TDM}~\cite{42}} & \parbox[t]{9cm}{\raggedright  
$\bullet$ Unbranched TDM-based probe used to study
remodeling enzymes such as Tdmh (TDM hydrolase) for \textit{in vitro}
and \textit{in vivo} assays \Lbreak (\textit{Msmeg}, \textit{Mtb})
\Lbreak $\bullet$ Probe also activated by hydrolases from other bacteria
species} \vspace*{2pt}\\\hline

{\raisebox{-1em}{\parbox[t]{3.2cm}{\raggedright  Bifunctional TMM chemical reporter}}}  &
\raisebox{-2em}{\textbf{\textit{O}-AzAlkTMM}~\cite{44}} & \parbox[t]{9cm}{\raggedright $\bullet$ First bifunctional
TMM-based probe \Lbreak $\bullet$ Simultaneously marks mycomembrane
biosynthesis by targeting mycoloyltransferases and subsequent trehalose
recycling  \Lbreak (\textit{Msmeg}, \textit{Mtb})}\vspace*{2pt}
\botline
\end{tabular}
\tabnote{$^a$Structures of the probes are shown in Figure~\ref{fig4}.}
\vspace*{-4pt}
\end{table*}

\section{Conclusion}\label{sec4}

In recent years, many smart probes have been designed and successfully
used in metabolic labeling experiments to explore the mycomembrane,
which is specific to mycobacteria and crucial for their survival. They
have provided new insights into the mycomembrane biogenesis as well as
potential new diagnostic tools for the detection of \textit{M.
tuberculosis}~\cite{5,6}. A summary of the main features of these
probes and their use is given in Tables~\ref{tab1} and~\ref{tab2} (see below). These
trehalose-based probes, based on the selective trehalose
functionalization, require important synthetic developments, especially
in glycochemistry, in order to obtain more and more efficient tools,
and we contributed to this exciting adventure. This review focused on
the chemical challenges for the synthesis of the trehalose- and
TMM-based probes described so far. Most of the reported trehalose-based
analogues are modified at C6. Indeed, selective reaction on primary
alcohols of trehalose is fast and convenient, and the $C_{2}$-symmetry
of trehalose makes the two primary positions equivalent. Modifications
at C2, C3 or C4 are more challenging and require more chemical steps to
retain an equatorial position on glucose unit. However, several studies
have shown a great tolerance of mycoloyltransferases.\unskip\break Indeed, the
synthesis of trehalose-based probes bearing the tag on axial position
of the carbohydrate ring could make faster the access to C2, C3 or C4
modified trehalose-based probes, by suppressing double inversion
protocols required to preserve the absolute configuration at C2, C3 or
C4. The second major chemical reporters are mimics of TMM. In this case
the same approach is used for most of the probes:  a selective
esterification on the same \unskip\break protected trehalose followed by acidic
deprotection. The main difficulty for the preparation of such TMM-based
probes is to synthesize the bioorthogonal functionalized mycolic acid
part of this glycolipid. Most of the reported tools are simplified
fatty acyl chain without ramification and $\upbeta$-OH group,
thus making their syntheses fast and efficient. In a recent work, our
group reported the first synthesis of a bioorthogonal TMM-based probes
with the natural pattern of mycolic acid which was an important
synthetic challenge. Interestingly, it proved to be very efficient in
labeling \textit{C. glutamicum} and such TMM-based probes may help
to elucidate the mycomembrane biogenesis at the molecular level.
Notably, they should help to decipher the enzymatic mechanism and
specificity of mycoloyltransferases. Altogether, the molecular tools
described here, in cooperation with biochemical approaches, could help
to better understand the role of all mycoloyltransferases involved in
the biogenesis of the mycomembrane, and may have a significant impact
in the characterization of new targets to develop innovative
therapeutic approaches.


\section*{Conflicts of interest}

Authors have no conflict of interest to declare.


\section*{Acknowledgments}
The authors thank the Minist\`{e}re de l'enseignement sup\'{e}rieur et
de la recherche for grants to EL and PR and for financial support. The
authors also thank the Agence Nationale de la Recherche (PTMyco, grant
NO. ANR-22-CE44-0005-03).

%\CDRGrant[ANR]{ANR-22-CE44-0005-03}

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