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\DOI{10.5802/crchim.356}
\datereceived{2024-09-27}
\daterevised{2024-10-21}
\dateaccepted{2024-10-21}
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\dateposted{2025-02-13}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{clarification}

\title{Synthesis of RNA conjugates as bisubstrates for the study of
transferases} 

\alttitle{Synth\`{e}se de conjugu\'{e}s d'ARN comme bisubstrats pour
l'\'{e}tude de transf\'{e}rases} 

\author{\firstname{Laura} \lastname{Iannazzo}\CDRorcid{0000-0002-2181-4885}}
\address{Universit\'{e} Paris Cit\'{e}, CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France}
\email[L. Iannazzo]{laura.iannazzo@u-paris.fr}

\author{\firstname{Emmanuelle} \lastname{Braud}\CDRorcid{0000-0002-2563-2675}}
\addressSameAs{1}{Universit\'{e} Paris Cit\'{e}, CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France}
\email[E. Braud]{emmanuelle.braud@u-paris.fr}

\author{\firstname{Matthieu} \lastname{Fonvielle}\CDRorcid{0000-0002-8013-8311}}
\address{Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Universit\'{e} Paris-Saclay, Gif-sur-Yvette, France}
\email[M. Fonvielle]{matthieu.fonvielle@i2bc.paris-saclay.fr}

\author{\firstname{M\'{e}lanie} \lastname{~Etheve-Quelquejeu}\CDRorcid{0000-0002-4105-3243}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Paris Cit\'{e}, CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France}
\address{Institut Universitaire de France (IUF), France}
\email[M. Etheve-Quelquejeu]{melanie.etheve-quelquejeu@u-paris.fr}

\shortrunauthors

\thanks{CNRS, Paris Cit\'{e} University, Agence Nationale de la
Recherche, La Ligue contre le Cancer}

\begin{abstract}
Bisubstrate compounds are designed to mimic the interaction of enzymes
with their two natural substrates. These molecules contain two moieties
mimicking the two substrates linked together by a covalent bridge, which
enhances both selectivity and potency. This review shows the
synthesis and utility of RNA conjugates, particularly peptidyl--RNA and
cofactor--RNA conjugates, as bisubstrate analogues for studying two
major families of RNA transferases: Fem aminoacyl transferases involved
in bacterial cell wall synthesis and m\tsup{6}A
methyltransferases, which catalyze RNA methylation. The synthetic
routes developed to access the RNA conjugates cover nucleoside and
nucleotide chemistry, site-specific RNA modifications, and solid phase
synthesis or chemoenzymatic processes as well as strategies of
oligonucleotide late-stage functionalization. The bisubstrate
molecules have been used not only as inhibitors of these transferases
but also as valuable chemical tools for elucidating enzyme mechanisms
and facilitating structural studies. 
\end{abstract}

\begin{altabstract}
Les compos\'{e}s bisubstrats sont con\c{c}us pour imiter
l'interaction entre les enzymes et leurs deux substrats naturels. Ces
mol\'{e}cules contiennent deux fragments qui miment les deux substrats
de l'enzyme, li\'{e}s entre eux par un lien covalent, ce qui permet
d'am\'{e}liorer \`{a} la fois la s\'{e}lectivit\'{e} et la puissance
des compos\'{e}s. Cette revue pr\'{e}sente la synth\`{e}se et
l'utilisation des conjugu\'{e}s d'ARN, en particulier des conjugu\'{e}s
peptidyl-ARN et cofacteur-ARN, comme analogues de bisubstrats pour
\'{e}tudier deux grandes familles d'ARN transf\'{e}rases: les
aminoacyl-transf\'{e}rases Fem impliqu\'{e}es dans la synth\`{e}se de
la paroi cellulaire bact\'{e}rienne et les m\tsup{6}A
m\'{e}thyltransf\'{e}rases qui catalysent la m\'{e}thylation de l'ARN.
Les voies de synth\`{e}se d\'{e}velopp\'{e}es pour acc\'{e}der aux
conjugu\'{e}s d'ARN font appel \`{a} la chimie des nucl\'{e}osides et
des nucl\'{e}otides, \`{a} des r\'{e}actions chimio sp\'{e}cifiques,
\`{a} la synth\`{e}se sur support solide, \`{a} des processus
chimio-enzymatiques, ou \`{a} des strat\'{e}gies de fonctionnalisation
tardive des oligonucl\'{e}otides. Les mol\'{e}cules bisubstrats ont
\'{e}t\'{e} utilis\'{e}es comme inhibiteurs des transf\'{e}rases, mais
\'{e}galement comme outils chimiques pour faciliter les \'{e}tudes
structurales et \'{e}lucider des m\'{e}canismes enzymatiques.
\end{altabstract}

\keywords{\kwd{Bisubstrate}
\kwd{Aminoacyl transferases}
\kwd{RNA methyltransferases}
\kwd{Nucleoside chemistry}
\kwd{RNA conjugates}}


\altkeywords{\kwd{Bisubstrat}
\kwd{aminoacyl-transf\'{e}rases}
\kwd{ARN m\'{e}thyltransf\'{e}rases}
\kwd{chimie des nucleosides}
\kwd{conjugu\'{e}s d'ARN}}


\maketitle

\vspace*{2pt}

\twocolumngrid

\end{noXML}


\section{Introduction} 

The bisubstrate strategy is recognized as a powerful approach for
developing inhibitors and chemical tools to study enzyme function. This
concept leverages the natural interaction of enzymes with multiple
substrates to design molecules that can simultaneously engage more than
one substrate-binding site. This approach often yields highly selective
and potent inhibitors, with selectivity arising from targeting two
distinct binding sites and potency enhanced by the combined binding
energies of both moieties and an entropy gain from a single molecule
interaction. By designing molecules that mimic the interactions of both
substrates, researchers can also create highly specific tools that
provide insights into the enzyme mechanism of action. This dual
engagement makes bisubstrate inhibitors valuable compounds for both
therapeutic development and fundamental research.


Based on this concept, the bisubstrate molecules contain two units
mimicking the two substrates attached together by a covalent linker
(Figure~\ref{fig1}). The linker, which is crucial for the correct
binding and \mbox{selectivity} of the bisubstrate, must be optimized based on
the distance between the two binding sites and the key residues
involved in the catalytic pocket.

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}Bisubstrate-binding mode.}
\end{figure}

RNA-dependent enzymes, particularly those involved in RNA modifications
or in non-ribosomal peptide synthesis, are especially amenable to
bisubstrate strategies~\cite{1}. In this account, we 
focus on the use of bisubstrate strategies to explore two families of
RNA-dependent enzymes: the Fem transferases and the
m\tsup{6}A methyltransferases. We first explain the
design of the bisubstrates, then describe their synthesis, and finally
their use to explore the mechanism of the reaction catalyzed by the
enzymes or as inhibitors of the transferases.


\section{Peptidyl--RNA conjugates for the study of Fem transferases} 

\subsection{The Fem transferases} 

Fem transferases are aminoacyl transferases that participate in
peptidoglycan synthesis in Gram-positive bacteria. They catalyze the
transfer of amino acids from aminoacyl-tRNA (aa-tRNA) to the amino
group of L-Lys of peptidoglycan precursors 
(Scheme~\ref{sch1})~\cite{2a,2b}. Enzymes of the Fem family are attractive 
targets for the development of antibiotics active against resistant
bacteria since the residues incorporated by the enzymes are essential
for the last cross-linking step of peptidoglycan polymerization in
several important ${\upbeta}$-lactam-resistant pathogens such as
staphylococci, pneumococci, and streptococci~\cite{3}.
Inhibiting Fem activity would therefore result in the production of
incomplete precursors acting as chain terminators that block the
formation of the essential peptidoglycan layer of the bacterial cell
wall\unskip\break \cite{4a,4b,4c}.

\begin{scheme*}
\vspace*{-.3pc}
\includegraphics{sc01}
\vspace*{6pt}
\caption{\label{sch1}Mechanism of the transfer of Ala from
Ala-tRNA$^{\mathrm{Ala}}$ to the amino group of Lys at the C-3
position of peptidoglycan precursors catalyzed by the aminoacyl
transferase FemX$_{\mathit{Wv}}$ from \textit{Weissella
viridescens}.}
\vspace*{-.4pc}
\end{scheme*}

In order to access chemical tools to achieve the resolution of the 3D
structure of complexes \mbox{comprising} the enzyme and the aa-tRNA or to
obtain inhibitors, the design and the synthesis of bisubstrates have
been described for two Fem enzymes: FemX$_{\mathit{Wv}}$~\cite{5a,5b} and 
FmhB~\cite{6} from \textit{Weissella viridescens} and
\textit{Staphylococcus aureus}, respectively. 



In \textit{Weissella viridescens}, the transferase FemX$_{\mathit{Wv}}$ uses
an ala-tRNA$^{\mathrm{ala}}$ to transfer an alanine to a
peptidoglycan fragment consisting of a pentapeptide tethered to a
uridine diphosphate \textit{N}-acetyl-muramic acid moiety~\cite{7}
(UDP--MurNAc) as described in Scheme~\ref{sch1}. 

To mimic the tetrahedral intermediate formed during the reaction
catalyzed by FemX$_{\mathit{Wv}}$, peptidyl--RNA conjugates were designed as
bisubstrates. These compounds contain double-stranded RNA of different
lengths simulating the natural substrate tRNA$^{\mathrm{ala}}$ and
a pentapeptide (L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala) tethered to a
UDP--MurNAc to mimic the growing peptidoglycan (Figure~\ref{fig2}).
Linkers based on 1,2,3-triazole and squaramide were chosen to link the two
units of the conjugates.


\begin{figure}
\includegraphics{fig02}
{\vspace*{-2pt}}
\caption{\label{fig2}General structure of peptidyl--RNA conjugates to mimic the
intermediate formed during the reaction catalyzed by
FemX$_{\mathit{Wv}}$.}
\end{figure}


\subsection{Synthesis of peptidyl--RNA conjugates}

\subsubsection{Peptidyl--RNA conjugates with a triazole linker}

\paragraph*{Synthesis of precursors}

Peptidyl--RNA conjugates containing a triazole linker as the bisubstrate of
Fem transferases were first synthesized by Fonvielle et~al.\ in
2013 using the Cu(I)-catalyzed azide--alkyne cycloaddition (CuAAC)
reaction~\cite{5a}. The chemical strategy relies on
the introduction of the azide function on the RNA moiety and the alkyne
on the peptide mimicking the growing peptidoglycan. 

The synthesis of  $2^\prime$-azido-RNA helixes was achieved using two
routes, either by solid phase synthesis (SPS) to obtain a series of
short azido-RNAs (18, 12, 10, and 8 nucleotides) or by a chemoenzymatic
approach to prepare azido-RNAs of different sizes simulating the
acceptor arm of the tRNA~\cite{5a}. In this study, 
$2^\prime$-azido-adenosine derivatives \textbf{7a} and \textbf{7b} were
synthesized in a seven-step procedure starting from commercially
available adenosine \textbf{1} (Scheme~\ref{sch2})~\cite{5a,5b}.

\begin{scheme*}
\includegraphics{sc02}
\vspace*{6pt}
\caption{\label{sch2}Synthesis of 
$2^\prime$-azido-$2^\prime$-deoxyadenosine \textbf{7a} and
\textbf{7b}.}
\vspace*{-8pt}
\end{scheme*}


To achieve the synthesis of the short double-stranded azido-RNAs by
SPS, $2^\prime$-azido-adenosine \textbf{7a} was first grafted on the resin
via a succinyl linker, the resulting adenosine \textbf{8}
being then engaged in SPS (Scheme~\ref{sch3}, Route A). The strategy
allows the introduction of a hexaethylene glycol linker to obtain
stabilized short double-stranded azido-RNAs~\cite{5b}.


\begin{scheme*}
\includegraphics{sc03}
\vspace*{6pt}
\caption{\label{sch3}The two routes for the synthesis of $2^\prime$-azido-RNAs. Route A:
synthesis of short azido-RNAs by SPS. Route B: synthesis of
azido-RNA by a chemoenzymatic approach. DMTr protecting group is
removed from the SPS machine.}
\end{scheme*}

The use of a chemoenzymatic strategy was also investigated by
Etheve-Quelquejeu and coworkers to provide azido-RNA with increased
size (Scheme~\ref{sch3}, route B). The key step of such an approach is
based on the synthesis of dinucleotide derivatives \textbf{12a--b} that
were obtained by the phosphoramidite coupling between compound
\textbf{7b} and the commercially available Ac-dC-PCNE and Ac-C-PCNE,
respectively. Since the T4 RNA ligase recognizes $5^\prime$-phosphorylated
RNAs, a chemical~\cite{2b} and an enzymatic phosphorylation~\cite{8} were
used to access $5^\prime$-phosphorylated $2^\prime$-azido-dinucleotides
\textbf{13a} and \textbf{13b}, respectively. Dinucleotides
\textbf{13a--b} were then ligated to a 22-nt RNA helix in the presence
of the T4 RNA ligase to produce the 24-nt azido-RNA helixes
\textbf{14a--b}. This chemoenzymatic strategy was also used to access
full tRNA analogues of 76 nucleotides with a sequence corresponding to
tRNA$^{\mathrm{Ala}}$~\cite{2b}, tRNA$^{\mathrm{Gly}}$~\cite{6},
and tRNA$^{\mathrm{Arg}}$~\cite{9}.

The second part of the bisubstrate structure, namely the peptide
mimicking the growing peptidoglycan, was synthesized by semi-synthesis
(Scheme~\ref{sch4}). To access the peptidoglycan precursor containing
an alkyne function \textbf{17}, \textit{meso}-cystine was enzymatically
incorporated into the peptidoglycan precursor providing compound
\textbf{15} (Scheme~\ref{sch4}). A reduction step in the presence of
dithiothreitol (DTT) followed by the conversion of the L-Cys into
dehydroalanine in the presence of
\textit{O}-(mesitylenesulfonyl)hydroxylamine (MSH) led to the formation
of UDP--MurNAc--pentapeptide \textbf{16}~\cite{5a}. Finally, the addition
of but-3-yne-1-thiol to the Michael acceptor produced 
alkyne-containing UDP--MurNAc--pentapeptide \textbf{17}. The formation of
the dehydroalanine into the peptide was optimized~\cite{4a,4b,4c} by using  
1,5-dibromohexanediamide instead of  MSH in the presence of
tris(2-carboxyethyl)phosphine (TCEP), providing a one-step process.
Worthy of note, compound \textbf{17} is a mixture of diastereoisomers
that were separated by HPLC purification.



\begin{scheme*}
\includegraphics{sc04}
\vspace*{6pt}
\caption{\label{sch4}Synthesis of a peptidoglycan fragment containing a propargyl group.}
\end{scheme*}

\vspace*{-2pt}

\paragraph*{Synthesis of peptidyl--RNA conjugates by CuAAC}

CuAAC is one of the most popular bioconjugation techniques reported in the past few
decades for the late-stage modification of biomolecules. In 2013, the
Etheve-Quelquejeu group reported the use of this reaction to access
peptidyl--RNA conjugates as bisubstrates of Fem transferases. 

The condition of the CuAAC reaction between $2^\prime$-azido-RNA \textbf{14a}
and alkyne UDP--MurNAc--pentapeptide \textbf{17} has to be  
optimized first. Using azido-RNA helix \textbf{14a} (50~mM), alkyne peptide
\textbf{17} (100~mM), copper sulfate (0.5~mM), sodium ascorbate (5~mM),
and tris[(1-hydroxypropyl-1\textit{H}-1,2,3-triazol-4-yl)methyl]amine
(THPTA) (3.5~mM) in water for 24~h at 37~\textdegree C led to the
expected peptidyl--RNA conjugate \textbf{18} in 36\% yield after
purification by denaturing polyacrylamide gel electrophoresis
(Scheme~\ref{sch5}A)~\cite{4a}. The THPTA ligand was required to
stabilize Cu(I) in aqueous buffer and avoid RNA degradation.

\begin{scheme*}
\includegraphics{sc05}
\vspace*{6pt}
\caption{\label{sch5}(A) The key CuAAC step to provide peptidyl--RNA conjugates to
study the transferase FemX$_{\mathit{Wv}}$ from \textit{Weissella
viridescens}. (B) Structure of the lipid--carbohydrate--peptidyl--RNA
conjugates synthesized by the same strategy to study the transferase
FmhB from \textit{Staphylococcus aureus}.}
\vspace*{-5pt}
\end{scheme*}

This versatile strategy was used in 2018 by Fonvielle et~al.\ 
to access even more complex conjugates, namely
lipid--carbohydrate--peptidyl--RNA conjugates \textbf{19} as bisubstrates
of the transferase FmhB (Scheme~\ref{sch5}B)~\cite{6}.

\subsubsection{Peptidyl--RNA conjugates with a squaramate linker}

In 2016, Fonvielle et~al.\ investigated the nature of the linker in
bisubstrate compounds using a squarate motif to tether the RNA
$3^\prime$-terminus and the peptide moiety. In this work, they described the
introduction of a squaramide linkage by the reaction of $2^\prime$-amino-RNAs
containing 4, 8, or 18 nucleotides with diethyl squarate diester
followed by the reaction of the amino group of the lysine of the
growing peptidoglycan peptide on the intermediate 
(Scheme~\ref{sch6})~\cite{10}. The synthesis starts with the reduction
of the azido group of compounds \textbf{20a--c} in the presence of TCEP
forming $2^\prime$-amino-RNA \textbf{21a--c}, which were then subjected to
1,4-addition with diethyl squarate diester to produce electrophilic RNA
\textbf{22a--c} with yields ranging from 75\% to 79\%.  
The reaction of RNAs
\textbf{22a--c} at pH 9.2 in the presence of 
UDP--MurNAc--pentapeptide \textbf{23} produced peptidyl--RNA conjugates
with a squaramate linker in 23\% to 45\% yield. Importantly, increasing
the size of the RNA moiety led to comparable reaction yields
(Scheme~\ref{sch6}). These results show that squarate-mediated
ligation provides a versatile route to peptidyl--RNA conjugates.
Significantly, this ligation is fully compatible with unprotected
UDP--MurNAc--pentapeptide, which contains reactive functions such as
phosphate, carbohydrate hydroxyl, and carboxyl groups.

\begin{scheme*}
\includegraphics{sc06}
\vspace*{6pt}
\caption{\label{sch6}Synthesis of peptidyl--RNA conjugates containing a squaramide linker.}
\vspace*{-8pt}
\end{scheme*}


\subsubsection{Synthesis of peptidyl--XNA conjugates}

In 2022, Rietmeyer et~al.\ explored the nature of the RNA by
reporting the synthesis of peptidyl--xeno-nucleic acid (XNA) conjugates
to evaluate the impact of the incorporation of XNA into the acceptor
arm of tRNA (Scheme~\ref{sch7})~\cite{11}. The bisubstrate molecules
described in this study were composed of the following: 
(1) a peptidyl part, mimicking the UDP--MurNAc--pentapeptide, one of
the substrates of Fem transferases; (2) an XNA part in which the
riboses of RNA were replaced by
$2^\prime$-deoxy-$2^\prime$-fluororibose,
$1^\prime$,$5^\prime$-anhydrohexitol, and
$2^\prime$-deoxy-$2^\prime$-fluoro-D-arabinose; and (3) a triazole
linker. A series of $2^\prime$-azido-XNA \textbf{25a--j} were prepared
and then subjected to CuAAC in the presence of alkyne
UDP--MurNAc--pentapeptide \textbf{17} using copper sulfate, sodium
ascorbate, and THPTA. Ten peptidyl--XNA 
conjugates \textbf{26a--j} were obtained under these reaction conditions
(Scheme~\ref{sch7}).



\begin{scheme*}
\includegraphics{sc07}
\vspace*{6pt}
\caption{\label{sch7}Synthesis of peptidyl--XNA conjugates.}
\vspace*{-.4pc}
\end{scheme*}


\subsection{Biological results} 

\subsubsection{Evaluation of peptidyl--RNA conjugates as\hfil\break inhibitors}

Triazole-containing peptidyl--RNA conjugates \textbf{12} and \textbf{13}
were tested as inhibitors of the Fem family of enzymes, demonstrating
picomolar inhibitory activity against the Fem transferases of
\textit{Weissella viridescens} and \textit{Staphylococcus aureus}, with
IC\tsub{50} values of $89 \pm 10~\mathrm{pM}$ against
FemX$_{\mathit{Wv}}$~\cite{5a} and $56 \pm 6~\mathrm{nM}$ against FmhB~\cite{6},
respectively. These results indicate that both components of the
peptidyl--RNA conjugates are required for potent inhibitory activity
since azido-RNA alone (compound \textbf{14a} in Scheme~\ref{sch3})
shows a $10^{4}$ fold higher IC\tsub{50} of $1.6 \pm
0.3~\upmu\mathrm{M}$~\cite{4b} compared to 89~pM.

Squaramide-containing peptide--RNA conjugates were also potent
inhibitors of FemX$_{\mathit{Wv}}$ though the triazole linker was clearly
preferred within the FemX$_{\mathit{Wv}}$ active site (IC\tsub{50} of
$123 \pm 6~\mathrm{nM}$ for the squaramide-containing conjugate
\textbf{24c} compared to $0.15 \pm 0.01~\mathrm{nM}$ for the
triazole-containing peptide--RNA conjugate \textbf{18}). In addition,
the squaramate unit also promoted specific cross-linking of RNA to the
catalytic lysine in FemX$_{\mathit{Wv}}$ but not to related transferases that
recognize different aminoacyl-tRNAs~\cite{10}.

The XNA conjugates were also evaluated as
inhibitors~\cite{11}. The findings reveal that XNA residues
located in the $3^\prime$-terminal single-stranded regions affect
ligand binding within the enzyme catalytic\break  site.


\subsubsection{Crystallographic studies}

The structure of FemX$_{\mathit{Wv}}$ was previously elucidated for both the
apo form and complexes containing UDP--MurNAc--pentapeptide or the
reaction product UDP--MurNAc--pentapeptide (Ala)~\cite{12}.
However, attempts to cocrystallize the enzyme with the RNA substrate
were unsuccessful. In contrast, triazole-containing peptidyl--RNA or
peptidyl--XNA conjugates enabled the successful crystallization of
FemX$_{\mathit{Wv}}$ in complex with these conjugates~\cite{5b,11}.

\begin{scheme*}
\vspace*{4pt}
\includegraphics{sc08}
\vspace*{8pt}
\caption{\label{sch8}Mechanism of the aminoacyl transfer catalyzed by
FemX$_{\mathit{Wv}}$~\cite{5b}.}
%\vspace*{4pt}
\end{scheme*}


In particular, the crystallographic structures of peptidyl--RNA
conjugates in complex with the {enzyme} provide insights into the enzyme
hot spots and catalytic mechanism. For instance, it has been
demonstrated that the tetrahedral intermediate formed during catalysis
is stabilized by Lys305 in FemX$_{\mathit{Wv}}$. This enzyme creates a unique
environment for substrate-assisted catalysis by orienting both
Ala-tRNA$^{\mathrm{Ala}}$ and UDP--MurNAc--pentapeptide into active
conformations for aminoacyl transfer. This mechanism is unique to
tRNA-dependent enzymes involved in protein-based peptide bond formation
(Scheme~\ref{sch8})~\cite{5b}.


Structural data from peptidyl--XNA conjugates also revealed, for the
first time, that the geometry of $1^\prime$,$5^\prime$-anhydrohexitol 
nucleotides closely resembles that of ribonucleotides, with the
$1^\prime$,$5^\prime$-anhydrohexitol of hexitol nucleic acids 
mimicking ribose in its C-3$^\prime$-endo sugar puckering. In
contrast, the incorporation of $2^\prime$-deoxy-$2^\prime$-fluoro-D-arabinose
nucleic acids ($2^\prime$F-ANA) and DNA residues into the
single-stranded region resulted in the loss of a ${\uppi}$-stacking
interaction, leading to misalignment of the ACCA terminal
moiety~\cite{11}.


\subsubsection{Conclusion}

Collectively, these examples highlight the potential of peptidyl--RNA
conjugates to explore non-ribosomal peptide synthesis, provide
structural insights into RNA--enzyme interactions, and develop potent
inhibitors.


\section{Cofactor--RNA conjugates for the study of m\tsup{6}A
methyltransferases}

\subsection{The m\tsup{6}A methyltransferases}

The S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTases)
catalyze the transfer of a methyl group to their substrate using mainly
the SAM cofactor as the methyl donor. This reaction
leads to the release of the coproduct S-adenosyl-L-homocysteine (SAH)
(Scheme~\ref{sch9}). The MTases methylate several types of substrates including
proteins, small molecules, and nucleic acids (DNA and RNA) on which they
are able to deposit this mark on different positions.


\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc09}
\vspace*{6pt}
\caption{\label{sch9}Methylation catalyzed by MTases.}
\vspace*{-3pt}
\end{scheme*}


The installation of a methyl group is the most frequent modification
found in RNA~\cite{13,14}. In particular, the introduction of
a methyl group on the exocyclic N-6 atom of adenosine
(m\tsup{6}A) by m\tsup{6}A MTases is the most
studied epitranscriptomic mark, identified and characterized across all
domains of life and all types of RNA~\cite{15}. The
m\tsup{6}A modification has been shown to be essential in
stabilizing RNA/RNA and RNA/protein
interactions~\cite{16,17}.

The m\tsup{6}A modification on mRNA is a dynamic and highly
regulated process, involving three distinct families of proteins, that is,
the writers (MTases) install the m\tsup{6}A mark, which is
recognized by the reader proteins and removed by the erasers
(Scheme~\ref{sch10}). m\tsup{6}A is the most abundant
internal modification in eukaryotic mRNA and long non-coding
RNA~\cite{18,19}. It regulates different aspects of RNA metabolism such
as splicing~\cite{20,21}, stability~\cite{22},
translation~\cite{23,24,25}, and many more biological
processes~\cite{26,27}.


Importantly, it has been shown that dysregulation of MTase expression
is correlated to human diseases including cancers~\cite{28,29,30}, type
2 diabetes~\cite{31}, neurological disorders~\cite{32}, cardiovascular
diseases~\cite{33}, and viral infections~\cite{34,35}.


\begin{scheme*}
\includegraphics{sc10}
\vspace*{6pt}
\caption{\label{sch10}The m\tsup{6}A modification process.}
\end{scheme*}

Accumulating evidence indicates that m\tsup{6}A MTases are
promising drug targets, and several pharmaceutical companies have
already developed inhibitors of the METTL3/METTL14 complex. For
example, STORM Therapeutics has announced the entry into Phase I of the
selective METTL3 inhibitor STC-15 for the treatment of acute myeloid
leukemia and solid tumors. An inhibitor of this enzyme complex
identified by a fragment-based approach has also been reported by
Gotham Therapeutics~\cite{36}. Very recently, the first
inhibitors of METTL16 have been also described~\cite{37}.


However, still little is known about the molecular mechanism of
m\tsup{6}A modification, including the recognition of the
RNA, which requires a sequence motif (DRACH, where A is the methylated
adenosine; D${=}$A, G, or U; R${=}$A or G; and H${=}$A, C, or U) and/or
RNA structure, the binding in the active site of the MTase, and finally
the release of the m\tsup{6}A substrate. This can be
explained by difficulties encountered in obtaining RNA--protein
crystallographic structures, leading to a limited number of structurally
characterized m\tsup{6}A MTases. To date, only the catalytic
domain of METTL16 has been crystallized with a substrate RNA~\cite{38},
and no structure of the ternary complex has been reported. The
structural studies carried out on human RNA MTases show that the
substrate-binding site is largely open on the 
SAM-binding pocket, favoring the design of bisubstrate
compounds~\cite{39}. In this context, bisubstrate analogues of MTases
designed to accommodate the SAM-binding domain and the 
substrate-binding site appear as important chemical tools to access
crystallographic structures that would help in  understanding better
the enzymatic reaction. These compounds are SAM--adenosine and
SAM--RNA conjugates, which contain an analogue of the cofactor SAM
covalently attached to a substrate surrogate via an
appropriate linker to mimic the transition state of the
SN\tsub{2} mechanism of the methylation reaction
(Figure~\ref{fig3}).


\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}General structure of SAM--RNA conjugates.}
\end{figure}


\vspace*{-2pt}

\subsection{Synthesis of SAM--adenosine and SAM--RNA conjugates}

\vspace*{-2pt}

In respect of the bisubstrate strategy, Atdjian et~al.\ reported in     
2018~\cite{40} a synthetic route to access SAM--adenosine conjugates.
The structures of these compounds contain a SAM analogue covalently
attached to a substrate surrogate via an alkyl linker that
mimics the transition state of the SN\tsub{2} mechanism of the
methylation. The key step of the synthesis is based on the concept of
the convertible nucleoside using the
$O^{6}$-(benzotriazol-1-yl)inosine~\cite{41a,41b,41c,41d} derivative as the 
electrophilic nucleoside to ensure the crucial connection between the
SAM moiety bearing an alkyl linker and the N-6
position of the adenosine.

Bisubstrate molecules containing a SAM analogue with an alkyl linker
\textbf{31a--b} were first synthesized (Scheme~\ref{sch11}) 
by Atdjian et~al.~\cite{40}.
Protected inosine \textbf{27} was activated in situ in the presence of
PyBOP and DBU in DMF to generate the corresponding activated inosine
intermediate, which was directly subjected to a nucleophilic aromatic
substitution (SN$_{\mathrm{Ar}}$) with $5^\prime$-aminoadenosine
derivatives \textbf{28a--b}, leading to the formation of 
SAM--adenosine conjugates \textbf{29a} and \textbf{29b} in 85\% yield.
The removal of the protecting groups was then achieved using
ZnBr\tsub{2} salt, yielding \textbf{30a} and \textbf{30b} in
71\% and 46\%, respectively. Treatment with ammonium fluoride gave
access to the fully deprotected bisubstrates \textbf{31a} and
\textbf{31b}.


\begin{scheme*}
\includegraphics{sc11}
\vspace*{8pt}
\caption{\label{sch11}Synthesis of adenosine--SAM bisubstrates containing an alkyl
linker by a convertible nucleoside approach.}
\end{scheme*}

To ensure better affinity with methyltransferase, a second
generation of SAM--adenosine conjugates with an alkyl linker was
prepared in which an ${\upalpha}$-amino-acid motif mimicking the
${\upalpha}$-amino-acid side chain of the cofactor SAM was
introduced~\cite{40}. The convertible nucleoside approach was
used in the presence of adenosine derivatives \textbf{32a--b}
substituted at the C-$5^\prime$ position by an ${\upalpha}$-amino-acid
residue and an alkyl linker containing two to three carbon atoms to access
compounds \textbf{33a} and \textbf{33b} in 80\% and 81\% yield,
respectively. A two-step procedure was applied to remove the protecting
groups of the adenosine analogues and the ${\upalpha}$-amino-acid side
chain, leading to the formation of bisubstrate molecules \textbf{35a}
and \textbf{35b} (Scheme~\ref{sch12}).


\begin{scheme*}
\includegraphics{sc12}
\vspace*{8pt}
\caption{\label{sch12}Synthesis of SAM--adenosine bisubstrates containing an alkyl
linker and ${\upalpha}$-amino-acid side chain.}
\vspace*{-1pt}
\end{scheme*}

To increase the size of the part mimicking the RNA substrate,
Etheve-Quelquejeu and coworkers applied the convertible nucleoside
strategy to short RNA strands~\cite{42}. Activated inosine \textbf{36}
was subjected to a phosphoramidite coupling in the presence of the
commercially available guanosine phosphoramidite and tetrazole in
acetonitrile. The triester phosphite intermediate was then oxidized with
a solution of diiodine in a water/THF/pyridine mixture to access the
corresponding phosphotriester. Deprotection of the C-5$^\prime$ position
was carried out by treatment with trichloroacetic acid in order to
remove the 4,4$^\prime$-dimethoxytrityl group. Dinucleotide
\textbf{37} was thus obtained in three steps in 56\% yield
(Scheme~\ref{sch13}). The SN$_{\mathrm{Ar}}$ reaction with SAM
analogue \textbf{32b} followed by three deprotection steps finally
yielded the dinucleotide \textbf{GA*}.

\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc13}
\vspace*{6pt}
\caption{\label{sch13}Synthesis of \textbf{GA*} dinucleotide--SAM conjugate.}
\vspace*{-5pt}
\end{scheme*}

The introduction of the SAM analogue in internal position requires the
preparation of the convertible phosphoramidite derivative \textbf{33},
in which the N-6 position is activated with an
$O^{6}$-(benzotriazol-1-yl) group in six steps starting from inosine
(Scheme~\ref{sch14}). The \textbf{A*A} dinucleotide-- and the
\textbf{GA*A} trinucleotide--SAM conjugates were obtained following the
phosphoramidite coupling strategy described above~\cite{42}.


\begin{scheme*}
\includegraphics{sc14}
\vspace*{6pt}
\caption{\label{sch14}Synthesis of convertible phosphoramidite \textbf{33} to access
SAM--\textbf{A*A} and SAM--\textbf{GA*A} conjugates.}
\vspace*{-.5pc}
\end{scheme*}

The synthesis of a SAM--RNA conjugate with a longer RNA strand was then
performed by a chemoenzymatic strategy~\cite{42}. An
enzymatic ligation catalyzed by the T4 RNA ligase was used to link a
dinucleotide covalently tethered to the SAM analogue (\textbf{pdGA*})
to a synthetic 11-nt RNA \mbox{molecule} to produce the corresponding 13-nt
SAM--RNA conjugate (Scheme~\ref{sch15}). 


\begin{scheme*}
\includegraphics{sc15}
\vspace*{6pt}
\caption{\label{sch15}Enzymatic ligation with T4 RNA ligase to access 13-nt SAM--RNA
conjugate.}
\vspace*{-2pt}
\end{scheme*}

Urea was also chosen to provide bisubstrates with a linker
containing only one carbon atom as in the SN\tsub{2} mechanism
of the methyl transfer catalyzed by the MTases~\cite{40}. To synthesize
such molecules, the exocyclic amine at the N-6 position of adenosine
\textbf{34} was first activated with \textit{iso}propenyl chloroformate
to access the corresponding \textit{iso}propenyl carbamate \textbf{35}
in 75\% yield (Scheme~\ref{sch16}). Then, the condensation with 
secondary amine \textbf{36} in pyridine produced urea \textbf{37} in
85\% yield. The deprotection of the silyl groups with
NH\tsub{4}F followed by the removal of the Boc groups and
\textit{tert}-butyl ester allowed the formation of urea bisubstrate
\textbf{38} in 47\% yield.


\begin{scheme*}
\includegraphics{sc16}
\vspace*{6pt}
\caption{\label{sch16}Synthesis of a SAM--adenosine conjugate with a urea
linker.}
\vspace*{-8pt}
\end{scheme*}


Moving forward to explore the impact of the linker of the SAM--adenosine
conjugates, Atdjian et~al.~\cite{43} examined in 2020 the CuAAC reaction
to introduce 1,4-disubstituted-triazole ring as a linker
(Scheme~\ref{sch17}). The CuAAC reaction between N-6-propargyl
adenosine \textbf{39} and $5^\prime$-azido-adenosine \textbf{40} was
conducted in the presence of sodium ascorbate and copper sulfate in
DMF/H\tsub{2}O to obtain triazole bisubstrate \textbf{41} in
36\% yield (Scheme~\ref{sch17}). Interestingly, this approach, allowing
the formation of a 1,2,3-triazole link between the adenosine and the
SAM analogue, does not require any protecting group on the reactive
functions of the nucleosides.


\begin{scheme*}
\vspace*{-2pt}
\includegraphics{sc17}
\vspace*{4pt}
\caption{\label{sch17}Synthesis of N-6 and N-1 SAM--adenosine conjugates by
CuAAC.}
\vspace*{-8pt}
\end{scheme*}


In addition to their effort to access the SAM--adenosine conjugate connected
at the N-6 position, Etheve-Quelquejeu and coworkers explored in the same
year~\cite{43} the reactivity of the N-1 position of
adenosine to prepare bisubstrate molecules to study
m\tsup{1}A MTase. The synthesis of SAM--adenosine conjugate
\textbf{43} with a triazole linker connected at the N-1 position was
achieved by the CuAAC between N-1 propargyl adenosine derivative
\textbf{42} and $5^\prime$-azido-adenosine \textbf{40}. In the presence of
sodium ascorbate and copper sulfate, the expected positively charged
conjugate \textbf{43} was isolated in 53\% yield (Scheme~\ref{sch17}). 

As seen above, CuAAC efficiently provides bisubstrate analogues with a
1,2,3-triazole linker. However, this strategy does not allow the
introduction of the ${\upalpha}$-amino-acid motif of the SAM cofactor.
To overcome this issue, Coelho et~al.\ developed in 2023 a two-step
metal-catalyzed procedure to access bisubstrate analogues of RNA
MTase~\cite{44}. In this work,  5-iodotriazole was modified
by metal-catalyzed reactions. The reactivity of adenosine derivatives
in iodo copper(I) azide--alkyne cycloaddition (iCuAAC) \mbox{reaction} was
achieved between adenosine \textbf{39} substituted at the N-6 position
with an alkyne function and $5^\prime$-azido-adenosine \textbf{40}.
The 5-iodotriazole compound \textbf{44} was obtained in 38\% yield in the
presence of a substoichiometric amount of
Cu(ClO\tsub{4})\tsub{2}, an excess of NaI as the
iodinating source, \textit{N},\textit{N}-diisopropylethylamine (DIPEA),
and tris(benzyltriazolylmethyl)amine  as the ligand in DMF
(Scheme~\ref{sch18}).



\begin{scheme*}
\includegraphics{sc18}
\vspace*{4pt}
\caption{\label{sch18}Synthesis of 5-iodotriazole compound \textbf{44} by iCuAAC.}
{\vspace*{-6pt}}
\end{scheme*}

A second metal-catalyzed promoted reaction was optimized to introduce
the ${\upalpha}$-amino-acid motif mimicking the methionine side chain
of the SAM cofactor~\cite{44}. First, a Sonogashira cross-coupling
\mbox{reaction} was examined between compound \textbf{44} and alkyne
\textbf{45} using Pd(OAc)\tsub{2}, CuI, triphenylphosphine
trisulfonate, and DIPEA in DMF to obtain the corresponding
coupling product in 13\% yield (Scheme~\ref{sch19}). The 
\textit{tert}-butyl and the Boc protecting groups were then removed
using ZnBr\tsub{2} salt in a mixture of \textit{iso}propanol and
nitromethane, producing compound \textbf{46} in 41\% yield. The
5-iodotriazole \textbf{44} is also a suitable substrate for the 
Suzuki--Miyaura cross-coupling reaction (Scheme~\ref{sch19}). In the
presence of pinacolboronic ester \textbf{47} with a water-soluble
palladium species in tris buffer, the corresponding cross-coupling
product was isolated in 57\% yield. A similar approach using the Stille
cross-coupling reaction in the presence of vinylstannane \textbf{48}
was reported to obtain the same analogue. A second step of deprotection
led to the formation of bisubstrate analogue \textbf{49} containing
a vinyl side chain in 47\% yield. \looseness=-1

\begin{scheme*}
\vspace*{-1pt}
\includegraphics{sc19}
\vspace*{6pt}
\caption{\label{sch19}Functionalization of 5-iodotriazole \textbf{44} by
metal-catalyzed cross-coupling.}
\vspace*{-3pt}
\end{scheme*}


The procedure developed by Coelho et~al.\ additionally allows access to
bisubstrate analogue \textbf{50} with an alkyl
chain~\cite{44}. A three-step procedure beginning with a
Suzuki--Miyaura cross-coupling reaction in the presence of alkyne
\textbf{47}, followed by the reduction of the double bond under
hydrogen atmosphere with PtO\tsub{2}, and a final deprotection
step of the ${\upalpha}$-amino-acid protecting groups provided compound
\textbf{50} in 40\% yield over two steps (Scheme~\ref{sch19}).


The two-step metal-catalyzed procedure developed by the group of
Etheve-Quelquejeu was next considered to synthesize bisubstrate
molecules with a longer RNA strand mimicking the substrate of the
methyltransferase (Scheme~\ref{sch20})~\cite{44}. This approach
requires the presence of an alkyne function at the internal position of
the oligonucleotide. This was achieved by the incorporation of a
phosphoramidite nucleoside containing an alkyne function at the N-6
position of the adenosine during oligonucleotide synthesis. The
resulting propargyl-N-6-A-RNA \textbf{51} was then subjected to iCuAAC
in the presence of $5^\prime$-azido-adenosine \textbf{52} to form
5-iodotriazole derivative \textbf{53}. A second step of late-stage
modification of the SAM-RNA conjugate, in the presence of
pinacolboronic ester \textbf{47}, followed by the removal of 
\mbox{\textit{tert}-butyl} and Boc protecting groups led to the formation of
bisubstrate analogue \textbf{54} containing a SAM analogue covalently
attached to the RNA substrate via a 1,2,3-triazole linker
substituted at the C-5 position with an ${\upalpha}$-amino-acid motif
(Scheme~\ref{sch20}).


\begin{scheme*}
\includegraphics{sc20}
\vspace*{6pt}
\caption{\label{sch20}Synthesis of SAM--RNA conjugate \textbf{54} with a triazole
linker substituted by an analogue of the methionine side
chain.}
\vspace*{-4pt}
\end{scheme*}


\subsection{Biological results} 

The SAM--adenosine conjugates were evaluated on the bacterial rRNA MTase
RlmJ, which methylates the 23S rRNA at position A2030 and on METTL3--14,
which deposits the m\tsup{6}A mark on human mRNA. This
enzymatic complex is involved in human diseases and considered  a
promising drug target~\cite{28,31,34}.

The binding of  SAM--adenosine conjugates \textbf{31a--b},
\textbf{35a--b}, and \textbf{38} to RlmJ was first assayed using
differential scanning fluorimetry~\cite{45}. Compounds
\textbf{31a--b} show an increase in the Tm value, indicating that they
establish stabilizing interactions with the protein compared to 
other potential bisubstrates. This result also underlines the
importance of the methionine part of the SAM analogue likely to be
involved in the interaction and an aliphatic linker. In addition,
compounds \textbf{35a} and \textbf{35b} display K$_{\mathrm{D}}$
values of 25 and $30~\upmu\mathrm{M}$, respectively. X-ray structures
were solved for the complexes \textbf{35a}/RlmJ and \textbf{35b}/RlmJ.
In the complex \textbf{35b}/RlmJ, one structure shows that the RNA
moiety of the conjugate binds the presumed substrate pocket of the
enzyme while the cofactor \mbox{analogue} occupies partly the SAM-binding
site, showing a rotation of 120\textdegree\ of the adenosine out of the
pocket (Figure~\ref{fig4}A). Importantly, compound \textbf{35b}
interacts with key residues involved in the catalytic mechanism, and the
crystallographic structure confirms the interactions of the methionine
part with the active site (Figure~\ref{fig4}B). All together, these
results indicate that the positioning of conjugate \textbf{35b} in RlmJ
resembles the transition state of the SN\tsub{2} mechanism of
the methyl transfer.


\begin{figure*}
\includegraphics{fig04}
\vspace*{-2pt}
\caption{\label{fig4}(A) Structure of \textbf{35b}/RlmJ (blue) aligned with
SAH/RlmJ (yellow)~\cite{45}. (B) Interaction map of
bisubstrate \textbf{35b} in the active site of
RlmJ~\cite{45}.}
\vspace*{-1pt}
\end{figure*}

In order to fully decipher the mechanism of methylation catalyzed by
METTL3--14,  conjugates \textbf{35a--b} were also used as tools in a
combined experimental and computational approach~\cite{46}. First, the
inhibitory potency of the compounds was determined using a reader-based
TR-FRET assay. Under these conditions, the conjugates exhibit
IC\tsub{50} values in the low micromolar range. Several
crystallographic structures of METTL3--14 in complex with compounds
\textbf{35a--b} were solved. Analysis of the interaction networks
between the active site and the conjugates validated their use as tools
to decipher the catalytic mechanism. In particular, compound
\textbf{35a} was identified as the transition state analogue of the methyl
transfer catalyzed by METTL3. Using the crystal structure of the
complex \textbf{35a}--METTL3--14 (Figure~\ref{fig5}), QM/MM simulations
allowed for the proposition of the mechanism catalyzed by METTL3--14.



\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig05}
\vspace*{-6pt}
\caption{\label{fig5}(A) Superposition of the crystal structure of METTL3--14 bound
to \textbf{35a} (magenta) and SAM (blue)~\cite{46}. (B)
Interaction map of bisubstrate \textbf{35a} in the active site of
METTL3--14~\cite{46}.}
\vspace*{-4pt}
\end{figure*}

To go further, SAM-RNA conjugates with increased size of the RNA part
were also used in crystallization assays with RlmJ to gain insights
into the~m\tsup{6}A methylation process~\cite{42}. Two
crystallographic structures were obtained with SAM-RNA conjugates
\textbf{GA*} and \textbf{GA*A} containing a dinucleotide and a
trinucleotide as the substrate, respectively. In the case of the
dinucleotide \textbf{GA*}, the adenosine of the SAM analogue shows a
correct orientation in the SAM pocket. All together, these two RX
structures allowed for the construction of a model showing the SAM
cofactor and a trinucleotide with the sequence
GA\tsup{2030}A of \textit{E.~coli} 23S rRNA bound to RlmJ.
Thanks to this model, we were able to propose a mechanism for the
methylation reaction catalyzed by RlmJ (Scheme~\ref{sch21})~\cite{42}.

\begin{scheme*}
\vspace*{-.2pc}
\includegraphics{sc21}
\vspace*{4pt}
\caption{\label{sch21}Proposed mechanism for the methylation reaction catalyzed by RlmJ~\cite{42}.}
\vspace*{-.8pc}
\end{scheme*}

In summary, the SAM--adenosine conjugates are valuable tools to decipher
the full mechanism of other RNA MTases and also for the design of
potent inhibitors.


\section{Conclusion and outlook}

{\vspace*{-2pt}}

The chemical approaches discussed in this review highlight the
versatility and efficiency of nucleoside and nucleotide chemistry in
addition to SPS and the chemoenzymatic approach in the
design of RNA conjugates for the study of RNA transferases. These
methods allow for significant flexibility in modifying the bisubstrate
compounds, including the RNA \mbox{sequence} and size, the nature of the
linkers, and the peptide or cofactor components. Such adaptability is
crucial for tailoring these chemical tools to explore a wide range of
transferases beyond the examples provided in this work, extending their
utility to new classes of RNA-dependent enzymes.

The insights they provide into enzyme--substrate interactions are
particularly valuable not only for fundamental research in RNA biology but also
for drug discovery. Indeed, since new transferases continue to be
discovered and considered as promising new drug
targets~\cite{47}, these chemical tools will be instrumental
in the development of new therapeutic strategies.

\section*{Declaration of interests}

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

\section*{Funding}

The authors thank the CNRS and Paris Cit\'{e} University for their
support and the Agence Nationale de la Recherche (ANR: ARNTools,
SyntRNA) and La Ligue contre le Cancer for funding.

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