# Synthesis of RNA conjugates as bisubstrates for the study of transferases

Laura Iannazzo, Emmanuelle Braud, Matthieu Fonvielle, Mélanie

Etheve-Quelquejeu

► To cite this version:

Laura Iannazzo, Emmanuelle Braud, Matthieu Fonvielle, Mélanie Etheve-Quelquejeu. Synthesis of RNA conjugates as bisubstrates for the study of transferases. Comptes Rendus. Chimie, 2025, 28 (G1), pp.111-129. 10.5802/crchim.356. hal-05287289

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# Comptes Rendus
Chimie

Laura Iannazzo, Emmanuelle Braud, Matthieu Fonvielle and Mélanie Etheve-Quelquejeu

Synthesis of RNA conjugates as bisubstrates for the study of transferases

Volume 1, issue 0 (0000), p. 000-000

https://doi.org/10.5802/crchim.356

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This article is licensed under the
CREATIVE COMMONS ATTRIBUTION 4.0 INTERNATIONAL LICENSE.
http://creativecommons.org/licenses/by/4.0/
Account

# Synthesis of RNA conjugates as bisubstrates for the study of transferases

Laura Iannazzo<sup>a</sup>, Emmanuelle Braud<sup>a</sup>, Matthieu Fonvielle<sup>b</sup> and Mélanie Etheve-Quelquejeu<sup>*,a</sup>

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<sup>a</sup> Université Paris Cité, CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France

<sup>b</sup> Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France

E-mails: laura.iannazzo@u-paris.fr (L. Iannazzo), emmanuelle.braud@u-paris.fr (E. Braud), matthieu.fonvielle@i2bc.paris-saclay.fr (M. Fonvielle), melanie.etheve-quelquejeu@u-paris.fr (M. Etheve-Quelquejeu)

**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<sup>6</sup>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.

**Keywords.** Bisubstrate, Aminoacyl transferases, RNA methyltransferases, Nucleoside chemistry, RNA conjugates.

**Funding.** CNRS, Paris Cité University, Agence Nationale de la Recherche, La Ligue contre le Cancer.

Manuscript received 27 September 2024, revised and accepted 21 October 2024.

## 1. 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 1). The linker, which is crucial for the correct binding and selectivity of the bisubstrate, must be optimized

<footnote>* Corresponding author</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Bisubstrate-binding mode.</figcaption></figure>

based on the distance between the two binding
sites and the key residues involved in the catalytic
pocket.

RNA-dependent enzymes, particularly those in-
volved in RNA modifications or in non-ribosomal
peptide synthesis, are especially amenable to bisub-
strate strategies [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<sup>6</sup>A methyltransferases. We first explain the de-
sign 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.

**2. Peptidyl–RNA conjugates for the study of Fem transferases**

**2.1. *The Fem transferases*

Fem transferases are aminoacyl transferases that par-
ticipate 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 1) [2,
3]. Enzymes of the Fem family are attractive tar-
gets 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 sev-
eral important β-lactam-resistant pathogens such as
staphylococci, pneumococci, and streptococci [4].
Inhibiting Fem activity would therefore result in the
production of incomplete precursors acting as chain
terminators that block the formation of the essen-
tial peptidoglycan layer of the bacterial cell wall
[5–7].

In order to access chemical tools to achieve
the resolution of the 3D structure of complexes

<figure><img src="image_4.png" /><figcaption>**Figure 2.** General structure of peptidyl–RNA conjugates to mimic the intermediate formed during the reaction catalyzed by FemX<sub>Wv</sub>.</figcaption></figure>

comprising the enzyme and the aa-tRNA or to obtain
inhibitors, the design and the synthesis of bisub-
strates have been described for two Fem enzymes:
FemX<sub>Wv</sub> [8,9] and FmhB [10] from *Weissella viri-
descens* and *Staphylococcus aureus*, respectively.

In *Weissella viridescens*, the transferase FemX<sub>Wv</sub> uses an ala-tRNA<sup>ala</sup> to transfer an alanine to a peptidoglycan fragment consisting of a pentapeptide tethered to a uridine diphosphate *N*-acetyl-muramic acid moiety [11] (UDP–MurNAc) as described in Scheme 1.

To mimic the tetrahedral intermediate formed
during the reaction catalyzed by FemX<sub>Wv</sub>, peptidyl-
RNA conjugates were designed as bisubstrates. These
compounds contain double-stranded RNA of differ-
ent lengths simulating the natural substrate tRNA<sup>ala</sup>
and a pentapeptide (L-Ala-D-iGlu-L-Lys-D-Ala-D-
Ala) tethered to a UDP–MurNAc to mimic the grow-
ing peptidoglycan (Figure 2). Linkers based on 1,2,3-
triazole and squaramide were chosen to link the two
units of the conjugates.

**2.2. Synthesis of peptidyl–RNA conjugates**

**2.2.1. Peptidyl–RNA conjugates with a triazole linker**

**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 [8]. The chemical strategy relies on the introduction of the azide function on
<figure><img src="image_5.png" /><figcaption>**Scheme 1.** Mechanism of the transfer of Ala from Ala-tRNA<sup>Ala</sup> to the amino group of Lys at the C-3 position of peptidoglycan precursors catalyzed by the aminoacyl transferase FemX<sub>Wv</sub> from *Weissella viridescens*.</figcaption></figure>

the RNA moiety and the alkyne on the peptide mimicking the growing peptidoglycan.

The synthesis of 2'-azido-RNA helices 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 [8]. In this study, 2'-azido-adenosine derivatives **7a** and **7b** were synthesized in a seven-step procedure starting from commercially available adenosine **1** (Scheme 2) [8,9].

To achieve the synthesis of the short double-stranded azido-RNAs by SPS, 2'-azido-adenosine **7a** was first grafted on the resin via a succinyl linker, the resulting adenosine **8** being then engaged in SPS (Scheme 3, Route A). The strategy allows the introduction of a hexaethylene glycol linker to obtain stabilized short double-stranded azido-RNAs [9].

The use of a chemoenzymatic strategy was also investigated by Etheve-Quelquejeu and coworkers to provide azido-RNA with increased size (Scheme 3, route B). The key step of such an approach is based on the synthesis of dinucleotide derivatives **12a-b** that were obtained by the phosphoramidite coupling between compound **7b** and the commercially available Ac-dC-PCNE and Ac-C-PCNE, respectively. Since the T4 RNA ligase recognizes 5'-phosphorylated RNAs, a chemical [3] and an enzymatic phosphorylation [12] were used to access 5'-phosphorylated 2'-azido-dinucleotides **13a** and **13b**, respectively. Dinucleotides **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 helices

**14a-b.** This chemoenzymatic strategy was also used to access full tRNA analogues of 76 nucleotides with a sequence corresponding to tRNA<sup>Ala</sup> [3], tRNA<sup>Gly</sup> [10], and tRNA<sup>Arg</sup> [13].

The second part of the bisubstrate structure, namely the peptide mimicking the growing peptidoglycan, was synthesized by semi-synthesis (Scheme 4). To access the peptidoglycan precursor containing an alkyne function **17**, mesocystine was enzymatically incorporated into the peptidoglycan precursor providing compound **15** (Scheme 4). A reduction step in the presence of dithiothreitol (DTT) followed by the conversion of the L-Cys into dehydroalanine in the presence of O-(mesitylenesulfonyl)hydroxylamine (MSH) led to the formation of UDP-MurNAc-pentapeptide **16** [8]. Finally, the addition of but-3-yne-1-thiol to the Michael acceptor produced alkyne-containing UDP-MurNAc-pentapeptide **17**. The formation of the dehydroalanine into the peptide was optimized [5-7] 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 **17** is a mixture of diastereoisomers that were separated by HPLC purification.

**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'-azido-RNA **14a** and alkyne UDP-MurNAc-

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Scheme 2.** Synthesis of 2'-azido-2'-deoxyadenosine 7a and 7b.</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Scheme 3.** The two routes for the synthesis of 2'-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.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Scheme 4.** Synthesis of a peptidoglycan fragment containing a propargyl group.</figcaption></figure>

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pentapeptide **17** has to be optimized first. Using azido-RNA helix **14a** (50 mM), alkyne peptide **17** (100 mM), copper sulfate (0.5 mM), sodium ascorbate (5 mM), and tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA) (3.5 mM) in water for 24 h at 37 °C led to the expected peptidyl-RNA conjugate **18** in 36% yield after purification by denaturing polyacrylamide gel electrophoresis (Scheme 5A) [4]. The THPTA ligand was required to stabilize Cu(I) in aqueous buffer and avoid RNA degradation.

This versatile strategy was used in 2018 by Fonvielle et al. to access even more complex conjugates, namely lipid-carbohydrate-peptidyl-RNA conjugates **19** as bisubstrates of the transferase FmhB (Scheme 5B) [10].

### 2.2.2. 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'-terminus and the peptide moiety. In this work, they described the introduction of a squaramide linkage by the reaction of 2'-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 6) [14]. The synthesis starts with the reduction of the azido group of compounds **20a-c** in the presence of TCEP forming 2'-amino-RNA **21a-c**, which were then subjected to 1,4-addition with diethyl squarate diester to

produce electrophilic RNA **22a-c** with yields ranging from 75% to 79%. The reaction of RNAs **22a-c** at pH 9.2 in the presence of UDP-MurNAc-pentapeptide **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 6). 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.

### 2.2.3. Synthesis of peptidyl-XNA conjugates

In 2022, Rietmeyer et al. explored the nature of the RNA by reporting the synthesis of peptidyl-xenonucleic acid (XNA) conjugates to evaluate the impact of the incorporation of XNA into the acceptor arm of tRNA (Scheme 7) [15]. 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'-deoxy-2'-fluororibose, 1',5'-anhydrohexitol, and 2'-deoxy-2'-fluoro-D-arabinose; and (3) a triazole linker. A series of 2'-azido-XNA **25a-j** were prepared and then subjected to CuAAC in the presence of alkyne UDP-MurNAc-pentapeptide **17** using copper sulfate, sodium ascorbate, and THPTA. Ten peptidyl-XNA conjugates **26a-j** were obtained under these reaction conditions (Scheme 7).
<figure><img src="image_11.png" /><figcaption>**Scheme 5.** (A) The key CuAAC step to provide peptidyl–RNA conjugates to study the transferase FemX<sub>Wv</sub> from *Weissella viridescens*. (B) Structure of the lipid–carbohydrate–peptidyl–RNA conjugates synthesized by the same strategy to study the transferase FmhB from *Staphylococcus aureus*.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Scheme 6.** Synthesis of peptidyl–RNA conjugates containing a squaramide linker.</figcaption></figure>

## 2.3. Biological results

### 2.3.1. Evaluation of peptidyl–RNA conjugates as inhibitors

Triazole-containing peptidyl–RNA conjugates **12** and **13** were tested as inhibitors of the Fem family of enzymes, demonstrating picomolar inhibitory activity against the Fem transferases of *Weissella viridescens* and *Staphylococcus aureus*, with IC<sub>50</sub> values of 89 ± 10 pM against FemX<sub>Wv</sub> [8] and 56 ± 6 nM against FmhB [10], respectively. These results indicate that both components of the peptidyl–RNA conjugates are required for potent inhibitory activity since azido-RNA alone (compound **14a** in Scheme 3) shows a 10<sup>4</sup> fold higher IC<sub>50</sub> of 1.6 ± 0.3 µM [6] compared to 89 pM.
<figure><img src="image_13.png" /><figcaption>**Scheme 7.** Synthesis of peptidyl-XNA conjugates.</figcaption></figure>

Squaramate-containing peptide-RNA conjugates were also potent inhibitors of FemX<sub>Wv</sub> though the triazole linker was clearly preferred within the FemX<sub>Wv</sub> active site (IC<sub>50</sub> of 123 ± 6 nM for the squaramide-containing conjugate **24c** compared to 0.15±0.01 nM for the triazole-containing peptide-RNA conjugate **18**). In addition, the squaramate unit also promoted specific cross-linking of RNA to the catalytic lysine in FemX<sub>Wv</sub> but not to related transferases that recognize different aminoacyl-tRNAs [14].

The XNA conjugates were also evaluated as inhibitors [15]. The findings reveal that XNA residues located in the 3'-terminal single-stranded regions affect ligand binding within the enzyme catalytic site.

### 2.3.2. *Crystallographic studies*

The structure of FemX<sub>Wv</sub> was previously elucidated for both the apo form and complexes containing UDP-MurNAc-pentapeptide or the reaction product UDP-MurNAc-pentapeptide (Ala) [16]. 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<sub>Wv</sub> in complex with these conjugates [9,15].

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

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demonstrated that the tetrahedral intermediate formed during catalysis is stabilized by Lys305 in FemX<sub>Wv</sub>. This enzyme creates a unique environment for substrate-assisted catalysis by orienting both Ala-tRNA<sup>Ala</sup> 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 8) [9].

Structural data from peptidyl-XNA conjugates also revealed, for the first time, that the geometry of 1',5'-anhydrohexitol nucleotides closely resembles that of ribonucleotides, with the 1',5'-anhydrohexitol of hexitol nucleic acids mimicking ribose in its C3'-endo sugar puckering. In contrast, the incorporation of 2'-deoxy-2'-fluoro-D-arabinose nucleic acids (2'F-ANA) and DNA residues into the single-stranded region resulted in the loss of a π-stacking interaction, leading to misalignment of the ACCA terminal moiety [15].

### 2.3.3. 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.

## 3. Cofactor-RNA conjugates for the study of m⁶A methyltransferases

### 3.1. *The m⁶A 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 9). 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.

The installation of a methyl group is the most frequent modification found in RNA [17,18]. In particular, the introduction of a methyl group on the exocyclic N-6 atom of adenosine (m⁶A) by m⁶A MTases is the most studied epitranscriptomic mark, identified and characterized across all domains of life and

all types of RNA [19]. The m⁶A modification has been shown to be essential in stabilizing RNA/RNA and RNA/protein interactions [20,21].

The m⁶A modification on mRNA is a dynamic and highly regulated process, involving three distinct families of proteins, that is, the writers (MTases) install the m⁶A mark, which is recognized by the reader proteins and removed by the erasers (Scheme 10). m⁶A is the most abundant internal modification in eukaryotic mRNA and long non-coding RNA [22,23]. It regulates different aspects of RNA metabolism such as splicing [24,25], stability [26], translation [27–29], and many more biological processes [30,31].

Importantly, it has been shown that dysregulation of MTase expression is correlated to human diseases including cancers [32–34], type 2 diabetes [35], neurological disorders [36], cardiovascular diseases [37], and viral infections [38,39].

Accumulating evidence indicates that m⁶A 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 [40]. Very recently, the first inhibitors of METTL16 have been also described [41].

However, still little is known about the molecular mechanism of m⁶A 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 m⁶A substrate. This can be explained by difficulties encountered in obtaining RNA-protein crystallographic structures, leading to a limited number of structurally characterized m⁶A MTases. To date, only the catalytic domain of METTL16 has been crystallized with a substrate RNA [42], 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 [43]. In this context, bisubstrate analogues of MTases designed to accommodate the SAM-binding domain and the
<figure><img src="image_14.png" /><figcaption>**Scheme 8.** Mechanism of the aminoacyl transfer catalyzed by FemX<sub>Wv</sub> [9].</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Scheme 9.** Methylation catalyzed by MTases.</figcaption></figure>

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<sub>2</sub> mechanism of the methylation reaction (Figure 3).

### 3.2. Synthesis of SAM-adenosine and SAM-RNA conjugates

In respect of the bisubstrate strategy, Atdjian et al. reported in 2018 [44] 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<sub>2</sub> mechanism of the
<figure><img src="image_16.png" /><figcaption>**Scheme 10.** The m⁶A modification process.</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 3.** General structure of SAM–RNA conjugates.</figcaption></figure>

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methylation. The key step of the synthesis is based on the concept of the convertible nucleoside using the O⁶-(benzotriazol-1-yl)inosine [45–48] 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 **31a–b** were first synthesized (Scheme 11) by Atdjian et al. [44]. Protected inosine **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<sub>Ar</sub>) with 5'-aminoadenosine derivatives **28a–b**, leading to the formation of SAM–adenosine conjugates **29a** and **29b** in 85% yield. The removal of the protecting groups was then achieved using ZnBr<sub>2</sub> salt, yielding

**30a** and **30b** in 71% and 46%, respectively. Treatment with ammonium fluoride gave access to the fully deprotected bisubstrates **31a** and **31b**.

To ensure better affinity with methyltransferase, a second generation of SAM–adenosine conjugates with an alkyl linker was prepared in which an α-amino-acid motif mimicking the α-amino-acid side chain of the cofactor SAM was introduced [44]. The convertible nucleoside approach was used in the presence of adenosine derivatives **32a–b** substituted at the C-5' position by an α-amino-acid residue and an alkyl linker containing two to three carbon atoms to access compounds **33a** and **33b** in 80% and 81% yield, respectively. A two-step procedure was applied to remove the protecting groups of the adenosine analogues and the α-amino-acid side chain, leading to the formation of bisubstrate molecules **35a** and **35b** (Scheme 12).

To increase the size of the part mimicking the RNA substrate, Etheve-Quelquejeu and coworkers applied the convertible nucleoside strategy to short RNA strands [49]. Activated inosine **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' position was carried out by treatment with trichloroacetic acid in order to remove the 4,4'-dimethoxytrityl group. Dinucleotide **37** was thus obtained in three steps in 56% yield (Scheme 13). The SN<sub>Ar</sub> reaction with SAM analogue **32b** followed
<figure><img src="image_18.png" /><figcaption>**Scheme 11.** Synthesis of adenosine–SAM bisubstrates containing an alkyl linker by a convertible nucleoside approach.</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Scheme 12.** Synthesis of SAM–adenosine bisubstrates containing an alkyl linker and α-amino-acid side chain.</figcaption></figure>

by three deprotection steps finally yielded the dinucleotide **GA**\*.

The introduction of the SAM analogue in internal position requires the preparation of the convertible phosphoramidite derivative **33**, in which the N-6 position is activated with an *O*\*6-(benzotriazol-1-yl) group in six steps starting from inosine (Scheme 14). The A\*A dinucleotide– and the GA\*A trinucleotide–

SAM conjugates were obtained following the phosphoramidite coupling strategy described above [49].

The synthesis of a SAM–RNA conjugate with a longer RNA strand was then performed by a chemoenzymatic strategy [49]. An enzymaticligation catalyzed by the T4 RNA ligase was used to link a dinucleotide covalently tethered to the SAM analogue (**pdGA**\*) to a synthetic 11-nt RNA molecule
<figure><img src="image_20.png" /><figcaption>**Scheme 13.** Synthesis of GA\* dinucleotide–SAM conjugate.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Scheme 14.** Synthesis of convertible phosphoramidite 33 to access SAM–A\*A and SAM–GA\*A conjugates.</figcaption></figure>

to produce the corresponding 13-nt SAM–RNA conjugate (Scheme 15).

Urea was also chosen to provide bisubstrates with a linker containing only one carbon atom as in the SN₂ mechanism of the methyl transfer catalyzed by the MTases [44]. To synthesize such molecules, the exocyclic amine at the N-6 position of adenosine **34** was first activated with isopropenyl chloroformate to access the corresponding isopropenyl carbamate **35** in 75% yield (Scheme 16). Then, the condensation with secondary amine **36** in pyridine produced urea **37** in 85% yield. The deprotection of the silyl groups with NH₄F followed by the removal of the Boc groups and tert-butyl ester allowed the formation of urea bisubstrate **38** in 47% yield.

Moving forward to explore the impact of the linker of the SAM–adenosine conjugates, Atdjian et al. [50] examined in 2020 the CuAAC reaction to introduce 1,4-disubstituted-triazole ring as a linker

(Scheme 17). The CuAAC reaction between N-6-propargyl adenosine **39** and 5'-azido-adenosine **40** was conducted in the presence of sodium ascorbate and copper sulfate in DMF/H₂O to obtain triazole bisubstrate **41** in 36% yield (Scheme 17). 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.

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 [50] the reactivity of the N-1 position of adenosine to prepare bisubstrate molecules to study m¹A MTase. The synthesis of SAM–adenosine conjugate **43** with a triazole linker connected at the N-1 position was achieved by the CuAAC between N-1 propargyl adenosine derivative **42** and 5'-azido-adenosine **40**. In the presence of sodium ascorbate
<figure><img src="image_22.png" /><figcaption>**Scheme 15.** Enzymatic ligation with T4 RNA ligase to access 13-nt SAM-RNA conjugate.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Scheme 16.** Synthesis of a SAM–adenosine conjugate with a urea linker.</figcaption></figure>

and copper sulfate, the expected positively charged conjugate **43** was isolated in 53% yield (Scheme 17).

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 α-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 [51]. In this work, 5-iodotriazole was modified by metal-catalyzed reactions. The reactivity of adenosine derivatives in iodo copper(I) azide–alkyne cycloaddition

(iCuAAC) reaction was achieved between adenosine **39** substituted at the N-6 position with an alkyne function and 5'-azido-adenosine **40**. The 5-iodotriazole compound **44** was obtained in 38% yield in the presence of a substoichiometric amount of Cu(ClO₄)₂, an excess of NaI as the iodinating source, N,N-diisopropylethylamine (DIPEA), and tris(benzyltriazolylmethyl)amine as the ligand in DMF (Scheme 18).

A second metal-catalyzed promoted reaction was optimized to introduce the α-amino-acid motif mimicking the methionine side chain of the SAM
<figure><img src="image_24.png" /><figcaption>**Scheme 17.** Synthesis of N-6 and N-1 SAM–adenosine conjugates by CuAAC.</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>**Scheme 18.** Synthesis of 5-iodotriazole compound 44 by iCuAAC.</figcaption></figure>

cofactor [51]. First, a Sonogashira cross-coupling reaction was examined between compound **44** and alkyne **45** using Pd(OAc)₂, CuI, triphenylphosphine trisulfonate, and DIPEA in DMF to obtain the corresponding coupling product in 13% yield (Scheme 19). The *tert*-butyl and the Boc protecting groups were then removed using ZnBr₂ salt in a mixture of isopropanol and nitromethane, producing compound **46** in 41% yield. The 5-iodotriazole **44** is also a suitable substrate for the Suzuki–Miyaura cross-coupling reaction (Scheme 19). In the presence of pinacolboronic ester **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 **48** was reported to obtain the same analogue. A second step of deprotection led to the formation of bisubstrate analogue **49** containing a vinyl side chain in 47% yield.

The procedure developed by Coelho et al. additionally allows access to bisubstrate analogue **50** with an alkyl chain [51]. A three-step procedure begin-

ning with a Suzuki–Miyaura cross-coupling reaction in the presence of alkyne **47**, followed by the reduction of the double bond under hydrogen atmosphere with PtO₂, and a final deprotection step of the α-amino-acid protecting groups provided compound **50** in 40% yield over two steps (Scheme 19).

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 20) [51]. 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 **51** was then subjected to iCuAAC in the presence of 5'-azido-adenosine **52** to form 5-iodotriazole derivative **53**. A second step of late-stage modification of the SAM-RNA conjugate, in the presence of pinacolboronic ester **47**, followed by the removal of
<figure><img src="image_26.png" /><figcaption>**Scheme 19.** Functionalization of 5-iodotriazole **44** by metal-catalyzed cross-coupling.</figcaption></figure>

tert-butyl and Boc protecting groups led to the formation of bisubstrate analogue **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 α-amino-acid motif (Scheme 20).

### 3.3. 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<sup>6</sup>A mark on human mRNA. This enzymatic complex is involved in human diseases and considered a promising drug target [32,35,38].

The binding of SAM–adenosine conjugates **31a–b**, **35a–b**, and **38** to RlmJ was first assayed using differential scanning fluorimetry [52]. Compounds **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 **35a** and **35b** display K<sub>D</sub> values of 25 and 30 µM, respectively. X-ray structures were solved for the complexes **35a/RlmJ** and **35b/RlmJ**. In the complex **35b/RlmJ**, one structure shows that the RNA moiety of the conjugate binds the presumed substrate pocket of the enzyme while the

cofactor analogue occupies partly the SAM-binding site, showing a rotation of 120° of the adenosine out of the pocket (Figure 4A). Importantly, compound **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 4B). All together, these results indicate that the positioning of conjugate **35b** in RlmJ resembles the transition state of the SN<sub>2</sub> mechanism of the methyl transfer.

In order to fully decipher the mechanism of methylation catalyzed by METTL3–14, conjugates **35a–b** were also used as tools in a combined experimental and computational approach [53]. First, the inhibitory potency of the compounds was determined using a reader-based TR-FRET assay. Under these conditions, the conjugates exhibit IC<sub>50</sub> values in the low micromolar range. Several crystallographic structures of METTL3–14 in complex with compounds **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 **35a** was identified as the transition state analogue of the methyl transfer catalyzed by METTL3. Using the crystal structure of the complex **35a–METTL3–14** (Figure 5), QM/MM simulations allowed for the proposition of the mechanism catalyzed by METTL3–14.
<figure><img src="image_27.png" /><figcaption>**Scheme 20.** Synthesis of SAM–RNA conjugate 54 with a triazole linker substituted by an analogue of the methionine side chain.</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 4.** (A) Structure of 35b/RlmJ (blue) aligned with SAH/RlmJ (yellow) [52]. (B) Interaction map of bisubstrate 35b in the active site of RlmJ [52].</figcaption></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 m⁶A methylation process [49]. Two crystallographic structures were obtained with SAM-RNA conjugates **GA*** and **GA***A containing a dinucleotide and a trinucleotide as the substrate, respectively. In the case of the dinucleotide **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 GA²⁰³⁰A of *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 21) [49].

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.
A

<figure><img src="image_29.png" /></figure>

B

<figure><img src="image_30.png" /><figcaption>**Figure 5.** (A) Superposition of the crystal structure of METTL3–14 bound to **35a** (magenta) and SAM (blue) [53]. (B) Interaction map of bisubstrate **35a** in the active site of METTL3–14 [53].</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>**Scheme 21.** Proposed mechanism for the methylation reaction catalyzed by RlmJ [49].</figcaption></figure>

**4. Conclusion and outlook**

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 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 [54], these chemical tools will be instrumental in the development of new therapeutic strategies.

**Declaration of interests**

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

The authors thank the CNRS and Paris Cité Université 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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Q1 Please provide orcid for all authors, if any.

Q2 'squaramide', 'squaramate' and 'square' are present. Please amend as appropriate.

Q3 The labeling of consecutive compounds in reactions is not consistent. For example, 7a and 7b, 12a-b, 13a and 13b, 13a-b, 28a-b, 29a and 29b, and so on are present. Please correct for consistency if necessary.

Q4 Ref [3a] changed to [4] as per reference list. Please check.

Q5 In Scheme 7 (yellow box, right-hand part), the green "DNA" conjugate appears of sensibly better quality than the other 4 conjugates: could you provide an improved file with all 5 conjugates in the same quality as "DNA" ?

We noted a spectacular difference in graphic quality between the docx and pdf versions of your manuscript (pdf much better with many figures of excellent vectorial quality). Therefore, we reverted to the figures from file CRChimie-Revised.pdf (uploaded October 17, 2024) rather than the final revised CRChimie-Revised2.docx (uploaded October 21 without a pdf counterpart). So it is possible that Figure 4B (currently of acceptable quality) could still be improved if you provided a file of vectorial quality."

Q6 'in complex with these conjugates' or 'forming complexes with these conjugates' or 'in the complexes formed with these conjugates' ?

Q7 'in complex with the enzyme' or 'forming complexes with the enzyme' or 'in the complexes formed with the enzyme'?

Q8 Both 'cofactor SAM' and 'SAM cofactor' are present. Please check and correct if necessary.

Q9 'N6' and 'N6' ('N-6 atom of adenosine') have been changed to 'N-6' to match other instances. Should 'C3' be changed to 'C-3' in 'C3'-endo sugar puckering'; 'C-3' is present in addition to 'C-5', 'C-5'' ('C-5' position) and 'N-1'.

Q10 Blue color 'A*' changed to black globally. Please confirm.

Q11 'in complex with compounds' or 'forming complexes with compounds' or 'in the complexes formed with compounds 35a-b'?

Q12 Please provide page range for Refs [2, 3].

Q13 Please provide volume number for Ref [44].

Q14 Please provide volume number page range/article number for Ref [53].

Q15 Multiple references are not allowed under the same key number. So we have split references [2, 4, 5] and [41] and renumbered the remaining references. Please check.