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# 1. Introduction

In eukaryotes and RNA viruses, RNA methylation is one of the most important post-transcriptional modifications of RNA, which is attracting growing interest in the field of epitranscriptomics, as it plays a crucial role in RNA stability, splicing, translation and as self-marker [1]. The methylation reaction is catalyzed by RNA methyltransferases (MTases) that use S-adenosyl-L-methionine (SAM) as a cofactor to transfer the methyl group to the 5'-terminal RNA cap or, internally, along the RNA sequence, releasing S-adenosyl-L-homocysteine (SAH). Methylation of ribonucleotides occurs at various positions on the nucleobase (⁷mG, m⁶A, m⁵C, m¹A...) or ribose moieties [2, 3]. In particular, the methylation of the 2'-hydroxyl of the nucleotide ribose (N<sub>m</sub>) is a ubiquitous epitranscriptomic modification often introduced at the first and second transcribed nucleotides downstream of the ⁷mG cap by 2'-O-methyltransferases (2'-O-MTases) giving rise to cap-1 (⁷mGpppN<sub>m</sub>) or cap-2 (⁷mGpppN<sub>m</sub>N<sub>m</sub>) [4]. 2'-O-methyl nucleotides have also been observed at internal sites of many RNA species, yet we have focused on 2'-O-methylation of the viral mRNA cap. This methyl decoration is important for RNA viruses to evade host innate immune responses by preventing recognition of their mRNAs as "non-self" by RIG like receptors as RIG-I and MDA5 [5-7]. Viral 2'-O-MTases therefore represent potential targets for antiviral drug development and for investigating unidentified molecular mechanisms of cap-1/2 formation, which underlies innate immunity evasion by viruses [8, 9]. It is noteworthy that even though most of 2'-O-MTases share a common Rossmann fold structural organization, the mechanism governing RNA recognition and their enzymatic activities varies among RNA viruses. For instance, in Flaviviruses (such as dengue (DENV) and Zika viruses (ZIKV)), the NS5 protein carries both N7- and 2'-O-MTase activities [10-13]. In contrast, coronaviruses encode two distinct enzymes: an N7-MTase in the C-terminal domain of nsp14 and a 2'-O-MTase on nsp16, which is regulated by a cofactor nsp10 [14-16]. A better understanding of how these proteins interact with their natural substrate and cofactor (mRNA and SAM) would enable to better know the environment of these MTases and design more potent inhibitors. Although several MTases have already been crystallized with or without the presence of a ligand (SAM, SAH, inhibitor) [17-19] or with cap analogs [11, 20], but to our knowledge only a limited set of MTases have been crystallized as ternary complexes with mRNA and SAM or SAH [21-24]. In some of these structures, the viral genome is outside the basic putative RNA binding groove, suggesting that the structure may represent a snapshot of the guanylylation reaction of the viral genome prior to its repositioning to reach the methyl donor sites [22]. Based on the results reported on RlmJ, a bacterial m⁶A rRNA MTase [25], we suggest that by designing short RNA covalently linked to a SAM analogue through the 2'-position of the 5'-terminal nucleotide (2'-O-methylation site), the interaction of the SAM mimetic with the protein could stabilize the RNA in the MTase methylation site, enabling its co-crystallization or its inhibition.
In a search for inhibitors of viral 2'-*O*-MTases and in contrast to existing small molecule inhibitors described for these targets [26-30], we chose to explore the promising bisubstrate approach with more complex molecules as a selective method of targeting MTases [31]. The bisubstrate molecules are designed as a representation of the transition state reached during the methylation reaction catalyzed by the MTase in the presence of the SAM methyl donor and the substrate [32]. Therefore, most RNA MTase bisubstrates consist of a SAM analogue linked to a molecule mimicking the RNA substrate. Recently, this approach has been used to target m<sup>6</sup>A MTases, valuable enzymes involved in a common post-transcriptional RNA modification with complex regulatory mechanisms [25, 33-36]. In these studies, a SAM analogue is connected *via* different linkers to the N6-atom of an adenosine within dinucleo(s)tides, a trinucleotide or a 13-mer RNA. As for our work, a similar bisubstrate approach was used to mimic the transition state of 2'-*O*-methylation of mRNA, with the design of dinucleosides with a 5'-OH adenosine in place of the first nucleotide downstream of the RNA cap (A1) linked from its 2' position to the 5' position of a second adenosine in place of the SAM adenosine (A2), *via* linkers of different sizes containing various heteroatoms (S, N) [31, 37]. Surprisingly, none of the synthesized 2',5'-linked dinucleosides showed inhibition of viral and human RNA 2'-*O*-MTases and, against all expectations, some displayed selective inhibition of nsp14, the N7-MTase of SARS-CoV [37]. The most plausible explanation for the lack of 2'-*O*-MTases inhibition of these dinucleosides is the absence of a cap structure which plays a key role for RNA recognition by RNA 2'-*O*-MTases. Indeed, the presence of a cap binding pocket allows the stacking of the GTP moieties by a phenylalanine residue (F25) in the NS5 2'-*O*-MTase of Zika or dengue flaviviruses [38, 39] and contributes to the ability of these MTases to recognize with high affinity 5'-cap mRNA (Gppp-mRNA) [10, 40], or specifically 5'-cap-0 mRNAs (<sup>7m</sup>Gppp-mRNA) as illustrated by nsp16 2'-*O*-MTase of SARS-CoV and SARS-CoV-2 [17, 24, 41].

We then hypothesized that short 5'-cap RNA structure inserting these 2',5'-linked dinucleosides might be good substrates for viral 2'-*O*-MTases, particularly those of dengue/Zika viruses and SARS-CoVs. To test this assumption, we report here the chemical synthesis on an automated DNA/RNA synthesizer of over 30 short oligoribonucleotides (ORNs) carrying various dinucleosides (X) in place of the 5'-terminal nucleotide of the RNA downstream of the cap. The sequences of the ORN-X conjugates prepared are composed of the first nucleotides of the viral RNA sequence of dengue/Zika viruses (5'-AGUUGU) [42], SARS-CoV (5'-AUAUUA) [43] or SARS-CoV-2 (5'-AUUAAA) [44]. In ORN-X conjugates, the first nucleotide downstream of the cap, which is naturally an adenosine, has been replaced by an adenine dinucleoside (X). Three series of capped or uncapped ORN-X of 4 or 6 nucleotides were prepared with at their 5'-end, different dinucleosides bearing a S-methylthioether linker (S-series, X<sup>S</sup>), N-ethylamino (X<sup>N</sup>) and N-ethyl-N-nosyl-amino (N<sup>NS</sup>) linkers (N-series) or a methyltriazole linker (Tz-series, X<sup>Tz</sup>) between the two adenosines A<sub>1</sub> and A<sub>2</sub> (Fig. 1). Some of the ORN-X synthesized were evaluated *in vitro* as potential
inhibitors of the 2'-*O*-MTase activity of NS5 proteins from dengue and Zika viruses and the nsp16/nsp10 protein complex from SARS-CoV-2. Remarkably, IC₅₀ values in the sub-µM range, suggesting a possible interaction of certain ORN conjugates with these viral 2'-*O*-MTases were confirmed by K_d values in the µM range with NS5 from dengue virus.

<figure><img src="image_1.png" /><figcaption>**Figure 1.** Schematic representation of ORN-X containing adenine dinucleosides (X) bearing S-methylthioether linker (ORN-X⁵) (S-series), N-ethylamino (ORN-X⁹) and N-ethyl-N-nosyl-amino linkers (ORN-X⁵Ns) (N-series) or a methyltriazole linker (ORN-X¹⁹) (Tz-series)</figcaption></figure>

## 2. Results and discussion

To synthesize ORN-X conjugates, two strategies were used for linker formation within dinucleosides X. Both S- and N-containing linkers between the two adenosines A₁ and A₂ were formed prior to ORN elongation on a solid support, by first preparing the corresponding 3'-phosphoramidite dinucleosides X⁵ 1 and X⁵Ns 2 (Schemes 1 and 2). Dinucleosides 1 and 2 were then incorporated into the 5'-end of the ORN chain, and the capping reaction was performed on solid-phase prior to deprotection of the ORN-X and its release from the support. Another strategy was used for the triazole-containing linker formed on a solid support, under copper-catalyzed azide-alkyne cycloaddition (CuAAC) conditions following ORN chain elongation and capping processes.

### 2.1. Synthesis of ORN-X via dinucleoside phosphoramidite incorporation (for S-series and N-series)

#### 2.1.1. Synthesis of dinucleoside phosphoramidites X⁵ 1 and X⁵Ns 2

The 3'-*O*-(2-cyanoethyl-*N*,*N*-diisopropylphosphoramidite)phosphoramidite 1 of the adenine dinucleoside 2',5'-bridged by a sulfur-containing linker was obtained in 5 steps from the previously
described S-dinucleoside **3** protected by a 5',3'-*O*-tetraisopropyldisiloxane group on A₁ (Schema 1) [31]. First, compound **3** was subjected to selective 2',3'-*O*-acetylation with acetic anhydride at 0°C to give **4** in 98% yield [45]. The reaction at 0°C prevents acetylation of the nucleobases. Next, the exocyclic N6 amino functions of adenines were protected by a phenoxyacetyl (Pac) group with Pac anhydride at 25°C in pyridine/THF [46]. We noted here that no reaction occurred in pyridine alone. Resulting compound **5** obtained in 78% yield was desilylated with a fluoride treatment to afford dinucleoside **6** with 5',3'-OH in A₁ (80% yield) [47]. A dimethoxytrityl group was then introduced onto the 5'-OH in 94% yield. The reaction was performed at 60°C as attempts at room temperature and even at 40°C failed. Finally, the resulting dinucleoside **7** was subjected to phosphitylation with 2-cyanoethyl N,N'-diisopropylchlorophosphoramidite to give rise to the desired dinucleoside phosphoramidite **1** in 43% yield contaminated with its oxidation product **8** in a 50/50 mixture as determined by <sup>31</sup>P-NMR [48]. As phosphoramidate **8** is not reactive during the coupling step of ORN elongation on solid support, the mixture of **1** and **8** was used as is for ORN-X<sup>S</sup> synthesis.

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Ratio 1:1</figcaption></figure>

**Scheme 1.** Synthesis of dinucleoside phosphoramidite X<sup>S</sup> **1**.

**Reagents and conditions:** (a) Ac₂O, pyridine, 0 °C, 3 h, 98%; (b) Pac₂O, pyridine/THF, 25°C, 15 h, 74%; (c) Et₃N.3HF, THF, 40°C, 17 h, 80%; (d) DMTrCl, pyridine, 60°C, 4 h, 94%; (e) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, DCM, 25°C, 80 min, 43%.

The dinucleoside 3'-*O*-phosphoramidite **2** with an ethylamino linker between the two adenosines was prepared from the previously described 5',3'-di-*O*-TBS-2'-*O*--(tosylethyl) adenosine **9** [31] and from 5'-*O*-tosyl-2,3'-di-*O*-acetyl adenosine **10** obtained in 80% yield from 5'-*O*-tosyl adenosine **11** by reaction with acetic anhydride (Scheme 2). Reaction in basic medium between **9** and 4-nitrobenzenesulfonamide led to protection of the 2'-*O*-ethylamino substituent of nucleoside **12** by a

<footnote>Ac = acetyl; Pac = phenoxyacetyl; iPr = isopropyl</footnote>
nosyl group in 74% yield [49]. Basic nucleophilic substitution of tosylate **10** by sulfonamide **12** creates the *N*-nosyl ethylamino internucleoside bond between A₁ and A₂ of **13** in 56% yield. As with phosphoramidite **1**, the exocyclic amino functions of the adenines of **13** were protected by phenoxyacetyl (Pac) groups in pyridine/THF to give **14** (76% yield), then the silyl protections were removed from A₁ to afford 5',3'-OH dinucleoside **15** (81% yield). DMTr protection was added at 60°C to the 5'-OH of **15** to give **16** (88% yield), which was then 3'-*O*-phosphitylated leading to phosphoramidite **2** (56% yield).

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

9

a

<figure><img src="image_9.png" /></figure>

12

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<figure><img src="image_11.png" /></figure>

11, R = H

10, R = OAc

<figure><img src="image_12.png" /></figure>

12

c

<figure><img src="image_13.png" /></figure>

13

d

<figure><img src="image_14.png" /></figure>

14

<figure><img src="image_15.png" /></figure>

15

f

<figure><img src="image_16.png" /></figure>

16

g

<figure><img src="image_17.png" /></figure>

2

TBS = tert-butyldimethylsilyl; Ac = acetyl; Pac = phenoxyacetyl; Ts = tosyl; Ns =nosyl; iPr = isopropyl

<caption>**Scheme 2.** Synthesis of dinucleoside phosphoramidite X<sup>Ns</sup> 2.</caption>

**Reagents and conditions:** (a) 4-nitrobenzenesulfonamide, KI, K₂CO₃, DMF, 50°C, 24 h, 74%; (b) Ac₂O, pyridine, 0°C, 3 h, 80%; (c) **10**, KI, K₂CO₃, DMF, 50°C, 24 h, 56%; (d) Pac₂O, pyridine/THF, 25°C, 24 h, 76%; (e) Et₃N·3HF, THF, 40°C, 24 h, 81%; (f) DMTrCl, pyridine, 60°C, 4 h, 88%; (g) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, DCM, 25°C, 90 min, 56%.

### 2.1.2. Synthesis of ORN-X<sup>S</sup> and ORN-X<sup>Ns</sup> conjugates

Dinucleoside phosphoramidites **1** and **2** were incorporated into 4-mer and 6-mer oligoribonucleotides using the same coupling conditions (180 s) as for the other ribonucleoside phosphoramidites, according to the 1 µmole solid-support oligoribonucleotide synthesis described by us [50]. The 5'-OH ORN-X and Gppp-ORN-X of the S- and N-series were synthesized using A, G and U ribonucleoside phosphoramidites protected by 2'-*O*-pivaloyloxymethyl (PivOM) groups (from Chemgenes, USA) (Schemes 3 and 4) [50]. To obtain 7mGpppORN-X, the PivOM phosphoramidite synthons were replaced by 2'-*O*-propionyloxymethyl (PrOM) ribonucleoside phosphoramidites, which allow the use of milder basic deprotection conditions to preserve cap-0 integrity as we recently reported [51].

As **1** was contaminated with phosphoramidate compound **8**, the coupling step was repeated twice before regular oxidation. 5'-OH ORN-X with adenine dinucleosides containing the methylthio X<sup>S</sup> or the
N-nosyl ethylamino X<sup>Ns</sup> linker were then deprotected and cleaved from the solid support using two-step deprotection under basic conditions, i.e. DBU followed by aqueous ammonia at room temperature, conditions described for RNA synthesis following the PivOM strategy [50]. To obtain 5'-OH ORN containing the *N*-ethylamino adenine dinucleoside X<sup>N</sup>, additional treatment with thiophenol and DBU was applied to remove the nosyl group prior to basic treatment. Crude ORN-X were purified by ion-exchange HPLC, and pure ORN-X<sup>S</sup>, ORN-X<sup>N</sup> and ORN-X<sup>Ns</sup> conjugates were characterized by MALDI-TOF mass spectrometry. Note that MALDI-TOF analysis of ORN-X<sup>Ns</sup> shows, alongside the expected ion, lower mass ions corresponding to ions resulting from known mono- and di-photo deoxygenations of the nitro group occurring during the laser irradiation of the samples (see supplementary material) [52].

<figure><img src="image_18.png" /><figcaption>**Scheme 3.** On-column synthesis of 5'-OH ORN-X<sup>S</sup>, GpppORN-X<sup>S</sup> and <sup>7m</sup>GpppORN-X<sup>S</sup> conjugates with dinucleoside X<sup>S</sup> as a 5'-terminal nucleotide analogue.</figcaption></figure>

**Reagents and conditions:** (a) 1M DBU / MeCN, 25°C, 3 min; (b) for OH or GpppORN-X<sup>S</sup>: aq. NH<sub>3</sub> 40°C, 3 h; for <sup>7m</sup>GpppORN-X<sup>S</sup>: 7M NH<sub>3</sub> in MeOH, 40°C, 3 h; (c) diphenylphosphite / pyridine, 40°C, 35 min; (d) 0.1 M TEAB, pH 8, 40°C, 45 min; (e) imidazole, CBrCl<sub>3</sub>, BSA, Et<sub>3</sub>N, MeCN, 40°C, 1 h; (f) for GpppORN-X<sup>S</sup>: 5'-diphosphate guanosine tri-*n*-butylammonium salt, ZnCl<sub>2</sub> / DMF, 40°C, 18 h; for <sup>7m</sup>GpppORN-X<sup>S</sup>: 5'-diphosphate N<sub>7</sub>-methylguanosine tri-*n*-butylammonium salt, ZnCl<sub>2</sub> / DMF, 40°C, 18 h.

5'-Cap GpppORN-X and 5'-Cap-0 <sup>7m</sup>GpppORN-X were obtained using our overall chemical process previously described [51, 53]. The introduction of unmethylated and N<sub>7</sub>-methylated caps onto ORN-X was performed on solid support at the end of ORN elongation (Schemes 3 and 4). As N<sub>7</sub>-methylguanosine is unstable in basic media during ORN deprotection, the 2'-O-PivOM protecting
groups were replaced by more base-labile 2'-O-PrOM groups, and standard aqueous ammonia was replaced by 7M methanolic ammonia to release the ORN-X [51]. As with the preparation of 5'-OH ORN-XN, the capped ORN-XN required additional thiophenolate treatment upon deprotection of the capped ORN-XNs (Scheme 4). Finally, GpppORN-X were deprotected and cleaved from the solid support with DBU followed by aqueous ammonia treatment, while methanolic ammonia was used to yield 7mGpppORN-X.

<figure><img src="image_19.png" /><figcaption>**Scheme 4.** On-column synthesis of 5'-OH ORN-X, GpppORN-X and 7mGpppORN-X conjugates of the N-series with dinucleosides XNs and XN as 5'-terminal nucleotide analogues.</figcaption></figure>

**Reagents and conditions:** (a) only for ORN containing XN: PhSH / DBU /DMF, 40°C, 18 h; (b) 1M DBU / MeCN 25°C, 3 min; (c) for OH or GpppORN-X: aq. NH₃ 40°C, 3 h; for 7mGpppORN-X: 7M NH₃ / MeOH; (d) diphenylphosphite / pyridine, 40°C, 35 min; (e) 0.1M TEAB, pH 8, 40°C, 45 min; (f) imidazole, CBrCl₃, BSA, Et₃N, MeCN, 40°C, 1 h; (g) for GpppORN-X: 5'-diphosphate guanosine tri-*n*-butylammonium salt, ZnCl₂ / DMF, 40°C, 18 h; for 7mGpppORN-X: 5'-diphosphate N7-methylguanosine tri-*n*-butylammonium salt, ZnCl₂ / DMF, 40°C, 18 h.

## 2.2. Synthesis of ORN-X<sup>TZ</sup> conjugates with a methyltriazole linker

In the case of the Tz-series ORN, a different strategy was used to create the internucleoside linker between A₁ and A₂ (Scheme 5). Instead of preparing a dinucleoside with the methyltriazole linker and then introducing it into the ORN, the linker was formed at the end of ORN elongation on a solid support by CuAAC. A propargyl function was introduced into ORN *via* the use of the commercially available adenosine 2'-*O*-propargyl-3'-*O*-phosphoramidite 17. CuAAC between the 2'-*O*-propargyl function of adenosine A₁ in ORN and 5'-azido adenosine 18 or its adenine analogue 19 was carried out in the
presence of copper (II) and sodium ascorbate under microwave irradiation (Scheme 5) [54, 55]. Click chemistry was performed on the capped ORN on solid support and, as reported in the literature, the cap structure, particularly the ⁷mG motif remained stable under these conditions [56, 57]. Note that Ethève-Quelquejeu et al. have used a similar linker to connect the N6 or N1 position of an adenine bearing a propargyl group to the 5' position of another adenosine as a SAM mimic in order to obtain bisubstrate interfering with m⁶A and m¹A RNA MTases [35, 36].

<figure><img src="image_20.png" /><figcaption>**Schema 5.** Synthesis of 5'-OH ORN-X^Tz, GpppORN-X^Tz and ⁷mGpppORN-X^Tz conjugates with dinucleosides X^Tz and X^TzN6 containing N6-meta-chlorobenzyl adenosine, as 5'-terminal nucleotide analogues.</figcaption></figure>

**Reagents and conditions** : ; (a) diphenylphosphite / pyridine, 40°C, 35 min; (b) 0.1M TEAB, pH 8, 40°C, 45 min; (c) imidazole, CBrCl₃, BSA, Et₃N, MeCN, 40°C, 1 h; (d) for GpppORN: 5'-diphosphate guanosine tri-*n*-butylammonium salt, ZnCl₂/DMF, 40°C, 18 h; for ⁷mGpppORN: 5'-diphosphate N7-methylguanosine tri-*n*-butylammonium salt, ZnCl₂/DMF, 40°C, 18 h; (e) **18** or **19**, CuSO₄, sodium ascorbate, dioxane/H₂O, 65°C MW, 1 h; (f) 1M DBU / MeCN, 25°C, 3 min; (g) for 5'-OH ORN-X or GpppORN-X: aq.NH₃, 40°C, 3 h; for ⁷mGpppORN-X: 7M NH₃/MeOH.

For the ORN-X^Tz synthesis, 5'-Azido adenosine **18** was obtained from 2',3'-*O*-isopropylidene adenosine in two steps (Scheme S1). In the first step, the azido function was introduced in the presence of
diphenylphosphoryl azide (DPPA) followed by treatment with sodium azide, giving rise to nucleoside **20** [58]. Next, isopropylidene was removed in acidic medium to afford **18**.

Flavivirus NS5 proteins have a unique conserved cavity, known to accommodate RNA cap structure (by stacking interaction mediated by F25) close to the SAM binding site [59]. Benzyl groups, in particular the *meta*-chlorobenzyl substituent on the N6 exocyclic amine of SAH adenine, have been shown to accommodate this cavity and provide inhibitors with enhanced activity and selectivity towards dengue 2'-*O*-MTase [26]. In a similar way, the exocyclic adenine amine of the A2 unit of ORN-X<sup>Tz</sup> has been functionalized with a 3-chlorobenzyl group. To introduce the N6-modified adenosine into ORN-X<sup>Tz</sup>, a 4-step synthesis pathway was carried out from commercial 6-chloroadenosine to obtain the azide-containing nucleoside **19** (Scheme S2). This starting material was 2',3'-*O*-protected by an isopropylidene group to give **21**. Then, an aromatic electrophilic substitution reaction carried out in basic medium in the presence of 2-chlorobenzylamine enabled functionalization of the adenine base at the N6 position of compound **22**. Finally, as before, DPPA followed by sodium azide treatments generated **23** with a 5'-azido function. Finally, after acidic treatment, the nucleoside **19** of interest was obtained (Scheme S2).

In all, over 30 ORN-X conjugates **24-57** were synthesized with different 5'-ends (OH, cap or cap-0) and various linkers between the SAM adenosine mimics and the ORN (Table 1). Substantial quantities (usually in the hundreds of nmoles) of high purity ORN-X were obtained by robust and efficient synthetic methods applied in solid phase: insertion of a modified dinucleoside phosphoramidite during RNA elongation (for ORN-X<sup>S</sup> and ORN-X<sup>N</sup> or ORN-X<sup>Ns</sup>) or post-RNA elongation click chemistry with a CuAAC reaction (for ORN-X<sup>Tz</sup> and ORN-X<sup>TzN6</sup>). Both methods are equally effective when the 5' end of ORN-X is OH or Gppp (average yield of 30% and 15%, respectively) but when it comes to <sup>7m</sup>Gppp, it would appear that the overall yields of ORN-X<sup>Tz</sup> synthesis 8.5%) are lower than those obtained by the phosphoramidite route (18.6% average yield). However, the click chemistry is more convenient and rapid for readily obtaining the dinucleoside on solid support than the multi-step preparation of the dinucleoside phosphoramidites.

<table><caption>**Table 1**
List and data on all synthesized ORN-X conjugates.<sup>a</sup></caption><thead><tr><th>5'-end</th><th>N°</th><th>Sequence<br>5'-3'</th><th>Calc. m/z</th><th>Found m/z<sup>b</sup></th><th>Quantities<br>(nmol)<sup>d</sup></th></tr></thead><tbody><tr><td rowspan="4">OH</td><td>24</td><td>X<sup>S</sup>GUU</td><td>1519.10</td><td>1518.79</td><td>441</td></tr><tr><td>25</td><td>X<sup>S</sup>GUUGU</td><td>2170.47</td><td>2170.16</td><td>241</td></tr><tr><td>26</td><td>X<sup>S</sup>UUA</td><td>1503.10</td><td>1503.42</td><td>261</td></tr><tr><td>27</td><td>X<sup>S</sup>UUAAA</td><td>2161.51</td><td>2161.01</td><td>509</td></tr></tbody></table>
<table><tbody><tr><td rowspan="10">Gppp</td><td>28</td><td>X<sup>Ns</sup>UAU</td><td>1685.23</td><td>1685.71</td><td>350</td></tr><tr><td>29</td><td>X<sup>Ns</sup>UAUUA</td><td>2320.61</td><td>2320.57</td><td>302</td></tr><tr><td>30</td><td>X<sup>N</sup>UAUUA</td><td>2135.45</td><td>2135.24</td><td>172</td></tr><tr><td>31</td><td>X<sup>Tz</sup>GUU</td><td>1555.09</td><td>1555.06</td><td>343</td></tr><tr><td>32</td><td>X<sup>Tz</sup>GUUGU</td><td>2206.47</td><td>2206.23</td><td>274</td></tr><tr><td>33</td><td>X<sup>Tz</sup>UUA</td><td>1538.09</td><td>1538.23</td><td>291</td></tr><tr><td>34</td><td>X<sup>Tz</sup>UAUUA</td><td>2174.47</td><td>2174.09</td><td>291</td></tr><tr><td>35</td><td>X<sup>S</sup>GUU</td><td>2024.26</td><td>2024.65</td><td>84</td></tr><tr><td>36</td><td>X<sup>S</sup>GUUGU</td><td>2675.64</td><td>2675.22</td><td>121</td></tr><tr><td>37</td><td>X<sup>S</sup>GUUGU-Cy5</td><td>3208.26<sup>c</sup></td><td>3208.61<sup>c</sup></td><td>91</td></tr><tr><td>38</td><td>X<sup>S</sup>UUAAA</td><td>2666.68</td><td>2667.05</td><td>185</td></tr><tr><td>39</td><td>X<sup>N</sup>UAUUA</td><td>2641.63</td><td>2641.18</td><td>184</td></tr><tr><td>40</td><td>X<sup>Tz</sup>GUU</td><td>2059.25</td><td>2059.44</td><td>124</td></tr><tr><td>41</td><td>X<sup>Tz</sup>GUUGU</td><td>2710.63</td><td>2710.20</td><td>162</td></tr><tr><td>42</td><td>X<sup>TzN6</sup>GUU</td><td>2183.82</td><td>2183.54</td><td>140</td></tr><tr><td>43</td><td>X<sup>TzN6</sup>GUUGU</td><td>2835.19</td><td>2834.98</td><td>139</td></tr><tr><td>44</td><td>X<sup>Tz</sup>GUUGU-Cy5</td><td>3243.25<sup>c</sup></td><td>3243.76<sup>c</sup></td><td>31</td></tr><tr><td rowspan="10">7mGppp</td><td>45</td><td>X<sup>S</sup>GUU</td><td>2039.30</td><td>2039.37</td><td>153</td></tr><tr><td>46</td><td>X<sup>S</sup>GUUGU</td><td>2690.67</td><td>2690.84</td><td>152</td></tr><tr><td>47</td><td>X<sup>S</sup>GUUGU-Cy5</td><td>3222.28<sup>c</sup></td><td>3222.25<sup>c</sup></td><td>46</td></tr><tr><td>48</td><td>X<sup>S</sup>UUA</td><td>2022.29</td><td>2022.30</td><td>251</td></tr><tr><td>49</td><td>X<sup>S</sup>UUAAA</td><td>2680.71</td><td>2680.70</td><td>206</td></tr><tr><td>50</td><td>X<sup>S</sup>UUAAA-Cy5</td><td>3213.33<sup>c</sup></td><td>3213.79<sup>c</sup></td><td>65</td></tr><tr><td>51</td><td>X<sup>Ns</sup>UAU</td><td>2205.43</td><td>2205.24</td><td>168</td></tr><tr><td>52</td><td>X<sup>Ns</sup>UAUUA</td><td>2840.81</td><td>2841.15</td><td>193</td></tr><tr><td>53</td><td>X<sup>Tz</sup>GUU</td><td>2073.28</td><td>2073.11</td><td>73</td></tr><tr><td>54</td><td>X<sup>Tz</sup>GUUGU</td><td>2724.65</td><td>2724.77</td><td>47</td></tr><tr><td>55</td><td>X<sup>Tz</sup>UUA</td><td>2057.28</td><td>2057.04</td><td>139</td></tr><tr><td>56</td><td>X<sup>Tz</sup>UUAAA</td><td>2715.70</td><td>2715.55</td><td>105</td></tr><tr><td>57</td><td>X<sup>Tz</sup>UUAAA-Cy5</td><td>3248.32<sup>c</sup></td><td>3247.91<sup>c</sup></td><td>62</td></tr></tbody></table>

## 2.3. Dose-response testing of ORN-X<sup>S</sup>, ORN-X<sup>Tz</sup>, ORN-X<sup>Ns</sup> and ORN-X<sup>N</sup> conjugates against SARS-CoV-2 nsp16/nsp10 2'-O-MTase

<footnote>aORN synthesis scale: 1 µmole</footnote>

<footnote>bMALDI-TOF characterization in negative mode [M-H]⁻</footnote>

<footnote>cMALDI-TOF characterization in negative mode [M⁺-2H]⁻</footnote>

<footnote>dquantities of isolated compound after IEX-HPLC purification and desalting</footnote>
To evaluate the possible interaction of ORN-X conjugates with the SARS-CoV-2 2'-O-MTase, we tested some of them with the 5'-terminal RNA sequence of SARS-CoV-2 or -CoV to determine their ability to inhibit the 2'-O-MTase activity of the nsp16/nsp10 complex. Briefly, purified nsp16 2'-O-MTase protein was incubated together with nsp10 protein in the presence of synthetic 7mGpppAC₄, radiolabeled 3H-SAM and the ORN-X conjugates. The RNA substrates were next separated from 3H-SAM by diethylaminoethyl (DEAE) filter binding assay (FBA) and the amount of 3H-Met transferred onto the RNA substrates was evaluated by counting the radioactivity associated to filtermat. Dose-response inhibition was performed by pre-incubation of the proteins with increasing concentrations of the best compounds and the IC₅₀ values, defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity were determined by Hillslope curve fitting.

The selected conjugates differ in the 5'-extremity (OH, Gppp, 7mGppp) of the ORN-X and in the dinucleoside (X<sup>S</sup>, X<sup>Tz</sup>, X<sup>Ns</sup> and X<sup>N</sup>) (Table 2). First, when the 5'-end of the conjugates is OH (entries 1-2, 6) or Gppp (entry 3), no inhibition of 2'-O-MTase was observed, meaning that ORN-X **26-27, 33 and 38** are probably not suitable to accommodate the nsp16/nsp10 catalytic pocket. This result is consistent with the capping pathway process in coronaviruses, since 2'-O-methylation takes place on an already N7-methylated cap. Indeed, conjugates with 7mGppp and bearing dinucleosides X<sup>S</sup> (entries 4-5) or X<sup>Tz</sup> (entries 7-8) showed submicromolar inhibitory activity irrespective of ORN length, with slightly better inhibition for ORN-X<sup>Tz</sup> **55-56**. In contrast, conjugates with bulkier, longer Ns and N linkers are much less active in inhibiting 2'-O-MTase (entries 9-11). The Ns and N linkers with an ethyl group instead of a methyl group, and thus with an additional methylene, do not seem to be adequate mimics of the 2'-O-methylation transition state. Based on this observation, we decided not to use Ns- and N-linked ORN-X conjugates for the remainder of our study and to focus instead on the S- and Tz-linked ORN-X conjugates.

<caption>**Table 2**</caption>

IC₅₀ values of ORN-X<sup>S</sup>, ORN-X<sup>Tz</sup>, ORN-X<sup>Ns</sup> and ORN-X<sup>N</sup> with SARS-CoV-2 or CoV RNA sequence on nsp16/nsp10 2'-O-MTase of SARS-CoV-2

<table><thead><tr><th>Entry</th><th>Linker Series</th><th>5'-end</th><th>N°</th><th>ORN-X sequence<sup>a</sup><br>5'-3'</th><th>SARS-CoV-2 nsp16/nsp10<br>IC<sub>50</sub><sup>b</sup> (µM)</th></tr></thead><tbody><tr><td>1</td><td rowspan="5">S</td><td rowspan="2">OH</td><td>26</td><td>X<sup>S</sup>UUA</td><td>n.i<sup>c</sup></td></tr><tr><td>2</td><td>27</td><td>X<sup>S</sup>UUAAA</td><td>n.i<sup>c</sup></td></tr><tr><td>3</td><td>Gppp</td><td>38</td><td>X<sup>S</sup>UUAAA</td><td>n.i<sup>c</sup></td></tr><tr><td>4</td><td rowspan="2">7mGppp</td><td>48</td><td>X<sup>S</sup>UUA</td><td>0.580 ± 0.140</td></tr><tr><td>5</td><td>49</td><td>X<sup>S</sup>UUAAA</td><td>0.511 ± 0.044</td></tr><tr><td>6</td><td rowspan="3">Tz</td><td>OH</td><td>33</td><td>X<sup>Tz</sup>UUA</td><td>n.i<sup>b</sup></td></tr><tr><td>7</td><td rowspan="2">7mGppp</td><td>55</td><td>X<sup>Tz</sup>UUA</td><td>0.295 ± 0.020</td></tr><tr><td>8</td><td>56</td><td>X<sup>Tz</sup>UUAAA</td><td>0.172 ± 0.039</td></tr></tbody></table>
<table><tr><td>9</td><td rowspan="2">Ns</td><td rowspan="2">7mGppp</td><td>51</td><td>X<sup>Ns</sup>UAU</td><td>54.2 ± 26</td></tr><tr><td>10</td><td>52</td><td>X<sup>Ns</sup>UAUUA</td><td>35.6 ± 18</td></tr><tr><td>11</td><td rowspan="2">N</td><td rowspan="2">Gppp</td><td>39</td><td>X<sup>N</sup>UAUUA</td><td>10.5 ± 2</td></tr><tr><td>12</td><td colspan="3">Sinefungin</td><td>15 ± 0.59</td></tr></table>

a SARS-CoV-2 5'-terminal RNA sequence: AUUAAA; SARS-CoV 5'-terminal RNA sequence: AUAUUA

b concentration inhibiting MTase activity by 50%; mean value from three independent experiments. Dose-response determination by FBA. A) Increasing concentrations of compounds were incubated with 100 nM nsp16 (MTase) and 600 nM nsp10 (Co-factor) of SARS-CoV-2 in the reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl₂, 1.9 µM SAM and 0.1 µM ³H-SAM (Perkin Elmer)] in the presence of 0.7 µM synthetic 7mGpppAC₄. Reactions were incubated at 30°C during 60 min and the enzymatic activity was determined by FBA. B) The IC₅₀ values were normalized and fitted with Prism (GraphPad) using the following equation: Y=100/(1+((X/IC₅₀)⁵Hillslope)) (n=3; mean value ± SD).

c n.i: no inhibition detected at 10 µM of ORN-X conjugate

## 2.4. Dose-response testing of ORN-X<sup>S</sup> and ORN-X<sup>Tz</sup> conjugates against NS5MTase<sub>DV2</sub> and NS5MTase<sub>ZV</sub>

In the same way, ORN-X conjugates were evaluated with the dengue and Zika virus 2'-O-MTases by measuring the inhibition of the MTase activity of recombinant methyltransferase domain of the non-structural protein NS5 from DV serotype 2 (NS5 MTase<sub>DV2</sub>) and ZIKV. The NS5 MTase<sub>DV2</sub> and MTase<sub>ZV</sub> were pre-incubated with increasing concentrations of ORN-X, and the MTase activity was measured by a filter-binding assay (FBA) as previously described. The IC₅₀ of some ORN-X<sup>S</sup> and ORN-X<sup>Tz</sup> conjugates were deduced from the Hillslope curve-fitting (Table 3).

<caption>**Table 3**</caption>

IC₅₀ values of ORN-X<sup>S</sup> and ORN-X<sup>Tz</sup> with DENV/ZIKV RNA sequence on NS5 MTases of DV₂ and ZIKV, and on nsp16/nsp10 2'-O-MTase of SARS-CoV-2

<table><thead><tr><th>Entry</th><th>Linker Series</th><th>5'-end</th><th>N°</th><th>ORN-X sequence<sup>a</sup><br>5'-3'</th><th>dengue virus NS5<br>IC<sub>50</sub> (µM)<sup>b</sup></th><th>Zika virus NS5<br>IC<sub>50</sub> (µM)<sup>b</sup></th><th>SARS-CoV-2<br>nsp16/nsp10<br>IC<sub>50</sub> (µM)<sup>b</sup></th></tr></thead><tbody><tr><td>1</td><td rowspan="4">S</td><td rowspan="2">Gppp</td><td>35</td><td>X<sup>S</sup>GUU</td><td>1.18 ± 0.53</td><td>n.d<sup>d</sup></td><td>18.70 ± 0.795</td></tr><tr><td>2</td><td>36</td><td>X<sup>S</sup>GUUGU</td><td>0.152 ± 0.02</td><td>0.185 ± 0.019</td><td>17.53 ± 2.63</td></tr><tr><td>3</td><td rowspan="2">7mGppp</td><td>45</td><td>X<sup>S</sup>GUU</td><td>0.882 ± 0.132</td><td>n.d</td><td>8.50 ± 0</td></tr><tr><td>4</td><td>46</td><td>X<sup>S</sup>GUUGU</td><td>0.218 ± 0.107</td><td>n.d</td><td>5.10 ± 0.8</td></tr><tr><td>5</td><td rowspan="4">Tz</td><td rowspan="2">OH</td><td>32</td><td>X<sup>Tz</sup>GUUGU</td><td>n.i<sup>c</sup></td><td>n.d</td><td>n.d</td></tr><tr><td>6</td><td>40</td><td>X<sup>Tz</sup>GUU</td><td>0.276 ± 0.045</td><td>n.d</td><td>n.d</td></tr><tr><td>7</td><td rowspan="2">7mGppp</td><td>41</td><td>X<sup>Tz</sup>GUUGU</td><td>0.168 ± 0.055</td><td>0.237 ± 0.016</td><td>16.20 ± 6.9</td></tr><tr><td>8</td><td>53</td><td>X<sup>Tz</sup>GUU</td><td>0.344 ± 0.019</td><td>n.d</td><td>n.d</td></tr><tr><td>9</td><td rowspan="2">TzN6<br>Bn</td><td rowspan="2">Gppp</td><td>54</td><td>X<sup>Tz</sup>GUUGU</td><td>0.196 ± 0.030</td><td>n.d</td><td>n.d</td></tr><tr><td>10</td><td>42</td><td>X<sup>TzN6</sup>GUU</td><td>0.227 ± 0.017</td><td>n.d</td><td>n.d</td></tr><tr><td>11</td><td>TzN6<br>Bn</td><td>Gppp</td><td>43</td><td>X<sup>TzN6</sup>GUUGU</td><td>0.217 ± 0.080</td><td>0.113 ± 0.012</td><td>n.d</td></tr></tbody></table>

a dengue and Zika viruses 5'-terminal RNA sequence: AGUUGU

b concentration inhibiting MTase activity by 50%; mean value from three independent experiments

Dose-response determination by FBA. A) Increasing concentrations of compounds were incubated with 500 nM of NS5 MTase of DV₂ or ZIKV, or 100 nM nsp16 (MTase) and 600 nM nsp10 (Co-factor) of Sars-CoV-2, in the reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 µM SAM and 0.1 µM ³H-SAM (Perkin Elmer)] in the presence of 0.7 µM synthetic 7mGpppAC₄. Reactions were incubated at 30°C during 30 min and the enzymatic activity was determined by FBA. B) The IC₅₀ values were
normalized and fitted with Prism (GraphPad) using the following equation: Y=100/(1+((X/IC₅₀)^(Hillslope))) (n=3; mean value ± SD)

c n.i: no inhibition detected at 10 µM of ORN-X conjugate

d not determined

Similarly to the data obtained with nsp16/nsp10 as target, the ORN-X<sup>Tz</sup> conjugate **32** with a 5'-OH terminus (entry 5, Table 3) did not inhibit NS5MTase<sub>DV2</sub> confirming that the cap is required for the interplay with both viral 2'-*O*-MTases. On the other hand, whether the conjugates are capped with Gppp or <sup>7m</sup>Gppp barely influence the NS5 inhibition efficiency, since it remains in the submicromolar range for most compounds, whatever the S or Tz linker used in the ORN-X conjugate (entries 2 and 4; entries 7 and 9, for instance). This observation is also consistent with the cap methylation process previously described for dengue or Zika viruses, as the same NS5 enzyme catalyses both N7- and 2'-*O*-methylation activities and the 2'-*O*-methylation did not depend on the presence of N7-methylation of the RNA substrate [11, 12]. Interestingly, ORN-X<sup>TzN6</sup> **42** and **43** showed IC<sub>50</sub> values in the same submicromolar range, whereas with introduction of the N6-benzyl- modified nucleobase, we expected an increase in inhibitory activity reflecting a better interaction with NS5, as described in the literature [26].

It was previously reported that the 2'-*O*-MTase activity increases with RNA substrate length [59] and we observe here again that 6-nucleotide-long ORN-X<sup>S</sup> or ORN-X<sup>Tz</sup> conjugates (**36, 41, 46, 54**) showed more effective inhibitory activity (IC<sub>50</sub> ≈ 0.2 µM) than the corresponding 4-nucleotide-long ORN-X conjugates (**35, 40, 45, 53**). This significant inhibition seems to anticipate that these 6-mer ORN-X substrates engage a direct interaction with the RNA binding groove of NS5MTase<sub>DV2</sub>. The influence of ORN length is more pronounced on inhibitory activity of ORN-X<sup>S</sup> (entries 1-2 and 3-4) than on ORN-X<sup>Tz</sup> (entries 6-7 and 8-9). The difference is negligible between IC<sub>50</sub> of 4-mer and 6-mer ORN-X<sup>TzN6</sup> (entries 10-11).

Interestingly, the IC₅₀ values against NS5MTase<sub>ZV</sub> are in the same submicromolar range as those against NS5MTase<sub>DV2</sub>, meaning that the interaction in the RNA binding site would be quite similar as expected regarding the conserved structural organization of both proteins (entries 2, 7, 11). In addition, we assessed substrate sequence specificity by testing some ORN-X with the dengue sequence (**35-36, 41, 45-46**) against SARS-CoV-2 nsp16/nsp10 and their inhibitory activity of this 2'-*O*-MTase was 10-fold weaker than against NS5MTase<sub>DV2</sub>, showing that recognition and interaction with NS5 are indeed sequence-specific.

**2.5. Protein-compound binding analysis by Fluorescence polarization (FP)**

In order to compare the binding properties of the compounds towards the NS5MTase<sub>DV2</sub>, we next determined the affinity constant (K<sub>d</sub>) by fluorescence polarization (FP) assay [60, 61]. We thus functionalized at 3'-end with Cy5 the ORN-X conjugates (**37, 44** and **47**, Table 1) and we first
demonstrated that the addition of the fluorescent dye did not alter the inhibition of the MTase activity of our compounds by filter-binding assay (not shown). Each compound was subsequently incubated with increasing concentrations of NS5MTase<sub>DV2</sub> protein in the FP assay. The curves show that the protein induced an increase of the FP signal, confirming that NS5MTase<sub>DV2</sub> interacts with all the three compounds and the deduced apparent K<sub>d</sub> values were in the µM range (Table 4, Supporting information).

<table><caption>**Table 4**

Protein-compound binding affinity by fluorescence polarization</caption><thead><tr><th>Entry</th><th>Linker Series</th><th>5'-end</th><th>N°</th><th>ORN-X sequence<sup>a</sup><br>5'-3'</th><th>dengue virus NS5<br>K<sub>d</sub> (µM)<sup>b</sup></th></tr></thead><tbody><tr><td>1</td><td rowspan="2">S</td><td>Gppp</td><td>37</td><td>X<sup>S</sup>GUUGU-Cy5</td><td>1.215 ± 0.225</td></tr><tr><td>2</td><td><sup>7</sup>mGppp</td><td>47</td><td>X<sup>S</sup>GUUGU-Cy5</td><td>1.245 ± 0.035</td></tr><tr><td>3</td><td>Tz</td><td>Gppp</td><td>44</td><td>X<sup>Tz</sup>GUUGU-Cy5</td><td>1.117 ± 0.084</td></tr></tbody></table>

Given the interesting K<sub>d</sub> values, we attempted co-crystallizations, however soaking experiments yielded no results. A meticulous effort in co-crystallization may perhaps provide new structures shedding light on our understanding of the molecular mechanisms governing methylation reactions or MTase inhibitions.

### 3. Conclusion

In this study, we reported the synthesis of original bisubstrates for the viral 2' O-MTases of SARS-CoV-2, dengue and Zika viruses, involving an adenosine as SAM mimic linked to the 2'-OH of the first nucleotide of short (4 or 6 nt) 5'-cap RNA via different linkers of various lengths containing S or N heteroatoms, or a 1,2,3-triazole moiety. We proposed two strategies to synthesize these ORN-X conjugates either by the incorporation of a pre-formed dinucleoside phosphoramidite into the 5'-end of the solid-supported ORN chain, or by click chemistry achieved post-RNA elongation on solid-phase but out of the synthesizer. First, two 3'-phosphoramidite dinucleosides containing S- or N-linkers were prepared in good yields, following a multi-step synthetic pathway. On the other hand, the 1,2,3-triazole (Tz) linker was readily obtained by a copper-catalyzed alkyne-azide cycloaddition between a 2'-O-propargyl adenosine inserted at the 5'-end ORN chain and a 5'-azido adenosine. This approach seems convenient to implement and provides easy access to different conjugates. As the natural substrate of

<footnote><sup>a</sup> dengue virus 5'-terminal RNA sequence: AGUUGU</footnote>

<footnote><sup>b</sup> determination of protein-compound binding affinity by fluorescence polarization. A) Increasing concentrations of NS5MTase<sub>DV2</sub> protein were incubated with the compounds labelled at the 3'-end with Cy5 (<b>37</b>, <b>44</b> and <b>47</b>). B) The dissociation coefficient (K<sub>d</sub>) was calculated from the FP data by site-specific binding regression analysis with Hill slope curve fitting using the following equation: Y=Bmax*X<sup>h</sup>/(K<sub>d</sub>*X<sup>h</sup> + X<sup>h</sup>) (Prism GraphPad software).</footnote>
2'-*O*-MTases is a 5' cap RNA, a cap structure (Gppp or ⁷mGppp) was added to the ORN after elongation and before click reaction (in the case of Tz-containing conjugates). The synthetic methods were efficient to afford over hundred nanomoles of each ORN-X conjugate.

Interestingly, some conjugates synthesized with S or Tz-linkers have been shown to inhibit the 2'-*O*-MTase nsp16/nsp10 of SARS-CoV-2 or the NS5 of dengue virus with $IC_{50}$ in the submicromolar range, with high selectivity due to the specificity of ORN sequence correlated to each virus. These data suggest a sequence-specific recognition and interaction of ORN conjugates with these viral 2'-*O*-MTases.

Therefore, such ORN-X conjugates can be used for all 2'-*O*-MTases to probe their active site, by adapting the RNA sequence to the RNA substrate of the MTase under investigation. They will be useful tools to obtain structures of MTase / bisubstrate complexes to unravel the cap RNA methylation process. These short ORN inhibitors could be also considered as potential therapeutic agents to interfere with viral MTases involved either in the capping pathway or in 2'-*O*-methylation inner the viral RNA and thus interplay with viral replication. With this aim, further work is now needed to increase the affinity of the inhibitors and modify them to ensure their delivery into the cells and their stability, which is a bottleneck for unmodified oligonucleotides.

# 4. Experimental section

## 4.1. Materials and methods

$CH_3CN$, pyridine, DIEA were distilled from calcium hydride. All dry solvents and reagents were purchased from commercial suppliers (Acros, Alfa Aesar, Sigma Aldrich, TCI Chemicals) and were used without further purification. Thin-layer chromatography (TLC) analyses were carried out on silica plate 60 $F_{254}$. Purifications by column chromatography were performed using Biotage Isolera 1 system with Flash-Pure Cartridges from Buchi.

¹H NMR and ¹³C NMR spectra were recorded with proton decoupling at ambient temperature on the following spectrometers: Bruker Avance III (400, 500 or 600 MHz). Chemical shifts (δ) are quoted in parts per million (ppm) referenced to the appropriate residual solvent peak: $CDCl_3$ at 7.26 ppm and DMSO-d₆ at 2.50 ppm relative to TMS. 2D NMR experiments including COSY, HSQC and HMBC were achieved to confirm proton assignments. Coupling constants (J) are given in Hertz (Hz). Multiplicity are indicated by: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad), dd (doublet of doublet). HRMS analyses were obtained with electrospray ionization on a Q-TOF Micromass spectrometer or with a Waters Synapt G25 (Waters, SN: UEB205) spectrometer equipped with positive electrospray source ionization (ESI). The capillary voltage was set to 2 kV and the sampling cone voltage was set to 30 V.

Analytical and semi-preparative high-performance anion-exchange chromatographies were performed on a Thermoscientific Ultimate 3000 system equipped with a LPG3400RS pump or HPG3200BX pump, a DAD 3000 detector and a WPS-3000TBRS autosampler, column oven TCC-3000SD. IEX-HPLC was performed on DNAPac PA200 columns (4x250 mm, 9x250 mm, Thermo Scientific). The following HPLC solvent systems were used: 5% $CH_3CN$ in 25 mM Tris-HCl buffer pH 8 (eluent A) and 5%
CH₃CN containing 400 mM of NaClO₄ in 25 mM Tris-HCl buffer pH 8 (eluent B). Flow rates were 1 mL/min, 4 mL/min for analytical, semi-preparative purposes, respectively. UV detection was performed at 260 nm. Chromeleon software was used.

MALDI-TOF mass spectra were recorded on an AXIMA Assurance (Shimadzu Biotech, UK) equipped with 337 nm nitrogen laser, using a saturated solution of 2,4,6-trihydroxyacetophenone in a solution of acetonitrile/ammonium citrate (0.1 M) (1:1; v/v). The samples were mixed with the matrix in a 1:1 ratio (v/v) crystalized on 384-well stainless steel plate and analyzed. UV quantification of ORN-X was performed on a UV-1600 PC UV/visible spectrophotometer (VWR) by measuring absorbance at 260 nm and using quartz cuvettes.

## 4.2. Chemical syntheses

### 4.2.1. Synthesis of dinucleoside phosphoramidite X⁵ 1

#### S-(3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methyladenosyl)-5'-thio-2',3'-O-acetyl-adenosine 4

To a solution of S-(3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methyladenosyl)-5'-thioadenosine 3 (1.6 g, 1.99 mmol, 1.00 eq) in anhydrous pyridine (7.3 mL) was added acetic anhydride (560 µL, 5.57 mmol, 2.8 eq). The reaction mixture was stirred at 0°C for 3 h under argon. Then, solvent was removed under vacuum and the resulting residue was coevaporated three times with toluene (3 x 20 mL) to give 4 as a white solid (1.73 g, 1.95 mmol, 98%). R<sub>f</sub> = 0.23 (MeOH/DCM 5:95). <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>) δ 8.35 (s, 1H), 8.26 (s, 1H), 8.08 (s, 1H), 7.99 (s, 1H), 6.18 (br s, 4H), 6.15 (d, J = 5.9 Hz, 1H), 6.02 (s, 1H), 5.99 (t, J = 5.8 Hz, 1H), 5.63 (dd, J = 5.6, 4.0 Hz, 1H), 5.15 (d, J = 11.6 Hz, 1H), 5.01 (d, J = 11.6 Hz, 1H), 4.74 (dd, J = 9.3, 4.7 Hz, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.50 (ddd, J = 6.3, 5.1, 4.0 Hz, 1H), 4.23 (dd, J = 13.3, 1.7 Hz, 1H), 4.12 (dt, J = 9.4, 2.2 Hz, 1H), 4.01 (dd, J = 13.4, 2.6 Hz, 1H), 3.23 (dd, J = 14.1, 5.1 Hz, 1H), 3.17 (dd, J = 14.1, 6.3 Hz, 1H), 2.13 (s, 3H), 2.06 (s, 3H), 1.13 – 0.90 (m, 28H). <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>) δ 169.8, 169.5, 155.9, 155.7, 153.6, 153.1, 150.0, 149.2, 139.0, 138.6, 120.4, 120.3, 88.7, 86.1, 82.5, 81.7, 78.2, 73.8, 73.2, 73.0, 69.6, 59.9, 32.5, 20.8, 20.6, 17.6 – 12.8. HRMS (ESI+): m/z calcd for C<sub>37</sub>H<sub>57</sub>N<sub>10</sub>O<sub>10</sub>SSi<sub>2</sub> [M + H]<sup>+</sup>: 889.3513, found: 889.3540.

#### S-(3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methyl-N6-phenoxyacetyladenosyl)-5'-thio-N6-phenoxyacetyl-2',3'-O-acetyladenosine 5

To a solution of 4 (1.16 g, 1.31 mmol, 1.00 eq) in anhydrous THF (13 mL) was added Pac<sub>2</sub>O (1.87 g, 6.53 mmol, 5.00 eq) and pyridine (530 µL, 6.53 mmol, 5.00 eq). The reaction mixture was stirred at room temperature for 15 h under argon. After reaction completion, a saturated aqueous NaHCO<sub>3</sub> was added. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-2% MeOH in DCM) to give 5 as a white foam (1.12 g, 0.97 mmol, 74%). R<sub>f</sub> = 0.81 (MeOH/DCM 5:95). <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>) δ 9.41 (br s, 1H), 9.38 (br s, 1H), 8.80 (s, 1H), 8.75 (s, 1H), 8.31 (s, 1H), 8.22 (s, 1H), 7.40 – 7.30 (m, 4H), 7.12 – 6.96 (m, 6H), 6.19 (d, J = 5.7 Hz, 1H), 6.06 (s, 1H), 6.03 (t, J = 5.7 Hz, 1H), 5.65 (t, J = 5.0 Hz, 1H), 5.13 (d, J = 11.6 Hz, 1H), 5.01 (d, J = 11.6 Hz, 1H), 4.92 – 4.80 (m, 4H), 4.75 (dd, J = 9.4, 4.7 Hz, 1H), 4.68 (d, J = 4.5 Hz, 1H), 4.52 (dt, J = 6.8, 4.3 Hz, 1H), 4.23 (d, J = 13.3 Hz, 1H), 4.14 (dt, J = 9.2, 2.0 Hz, 1H), 4.02 (dd, J = 13.5, 2.5 Hz, 1H), 3.26 (dd, J = 14.3, 4.5 Hz, 1H), 3.14 (dd, J = 14.2, 6.7 Hz, 1H), 2.14 (s, 3H), 2.07 (s, 3H), 1.15 – 0.90 (m, 28H). <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>) δ 169.7, 169.3, 166.6 (2C), 157.0, 157.0, 152.9, 152.6, 151.6, 150.9, 148.6, 148.3, 142.0, 141.6, 129.9, 123.4, 123.3, 122.5, 122.5, 115.0, 115.0, 88.7, 86.4, 82.6, 81.7, 77.9,
73.8, 73.0, 72.7, 69.5, 68.2, 68.1, 59.6, 32.5, 20.6, 20.4, 17.4 - 12.6. HRMS (ESI+): m/z calcd for C₅₃H₆₉N₁₀O₁₄SSi₂ [M + H]⁺: 1157.4248, found: 1157.4249.

### S-(2'-O-methyl-N<sup>6</sup>-phenoxyacetyladenosyl)-5'-thio-N<sup>6</sup>-phenoxyacetyl-2',3'-O-acetyladenosine 6

To a solution of 5 (1.12 g, 0.97 mmol, 1.00 eq) in anhydrous THF (9.7 mL) was added Et₃N.3HF (950 µL, 5.81 mmol, 6.00 eq). The reaction mixture was stirred at 40°C for 17 h under argon. After reaction completion, TEAAc buffer 50 mM, pH 7 was added (5 mL). The aqueous layer was extracted with DCM and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-5% MeOH in DCM) to give 6 as a white foam (710 mg, 0.78 mmol, 80%). R_f = 0.34 (MeOH/DCM 5:95). <sup>1</sup>H NMR (600 MHz, CDCl₃) δ 9.58 (br s, 1H), 9.56 (br s, 1H), 8.79 (s, 1H), 8.74 (s, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 7.38 - 7.29 (m, 4H), 7.08 - 7.00 (m, 6H), 6.21 (d, J = 6.2 Hz, 1H), 6.15 (t, J = 5.9 Hz, 1H), 5.91 - 5.80 (m, 2H), 5.65 (dd, J = 5.6, 3.6 Hz, 1H), 4.94 - 4.85 (m, 4H), 4.83 (dd, J = 7.6, 4.5 Hz, 1H), 4.59 (d, J = 11.9 Hz, 1H), 4.57 - 4.50 (m, 2H), 4.34 - 4.24 (m, 2H), 3.94 (dt, J = 13.1, 1.8 Hz, 1H), 3.77 - 3.71 (m, 1H), 3.34 (d, J = 2.3 Hz, 1H), 3.10 - 2.97 (m, 2H), 2.16 (s, 3H), 2.06 (s, 3H). <sup>13</sup>C NMR (150 MHz, CDCl₃) δ 170.0, 169.8, 167.0 (2C), 157.2 (2C), 153.0, 152.1, 151.9, 150.8, 149.3, 148.8, 144.1, 142.8, 130.0, 124.3, 123.5, 122.6, 122.6, 115.1, 115.1, 89.1, 88.3, 86.4, 83.2, 81.4, 76.0, 72.7, 72.6, 71.0, 68.4, 68.3, 63.3, 35.0, 20.8, 20.5. HRMS (ESI+): m/z calcd for C₄₁H₄₃N₁₀O₁₃S [M + H]⁺: 915.2726, found: 915.2732.

### S-(5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-N<sup>6</sup>-phenoxyacetyladenosyl)-5'-thio-N<sup>6</sup>-phenoxyacetyl-2',3'-O-acetyladenosine 7

After three coevaporations of 6 with anhydrous pyridine (40 mL), a solution of 6 (715 mg, 0.78 mmol, 1.00 eq) in anhydrous pyridine (6.0 mL) was heated at 60°C under argon. Then, 4,4'-dimethoxytrityl chloride (449 mg, 1.33 mmol, 1.70 eq) was added in small portions over 30 min. The mixture was stirred for 4 h at 60°C. After cooling to room temperature, a saturated aqueous NaHCO₃ was added. The aqueous layer was extracted with DCM and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-2% MeOH in DCM, 1% pyridine) to give 7 as a beige foam (890 mg, 0.73 mmol, 94%). R_f = 0.35 (MeOH/DCM 2:98). <sup>1</sup>H NMR (600 MHz, CDCl₃) δ 9.45 (br s, 1H), 9.44 (br s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.63 - 8.58 (m, 3H), 8.25 (s, 1H), 8.21 (s, 1H), 7.70 - 7.64 (m, 1H), 7.46 - 7.39 (m, 2H), 7.37 - 7.17 (m, 7H), 7.09 - 7.00 (m, 6H), 6.83 - 6.76 (m, 4H), 6.16 (d, J = 5.9 Hz, 1H), 6.13 (d, J = 4.6 Hz, 1H), 6.03 (t, J = 5.8 Hz, 1H), 5.59 (dd, J = 5.7, 4.0 Hz, 1H), 4.94 - 4.79 (m, 7H), 4.51 (t, J = 4.9 Hz, 1H), 4.35 (dt, J = 7.0, 4.3 Hz, 1H), 4.21 (q, J = 4.1 Hz, 1H), 3.76 (s, 6H), 3.51 (dd, J = 10.7, 3.3 Hz, 1H), 3.39 (dd, J = 10.8, 4.1 Hz, 1H), 3.15 (br s, 1H), 3.08 (dd, J = 14.5, 4.3 Hz, 1H), 3.02 (dd, J = 14.4, 6.7 Hz, 1H), 2.13 (s, 3H), 2.03 (s, 3H). <sup>13</sup>C NMR (150 MHz, CDCl₃) δ 169.8, 169.5, 166.7 (2C), 158.6, 157.1, 157.0, 152.9, 152.6, 151.6, 151.5, 149.8, 149.8, 148.6, 148.4, 144.5, 142.3, 142.2, 136.0, 135.6, 135.6, 130.1, 129.9, 128.2, 127.9, 127.0, 123.7, 123.3, 123.2, 122.4 (2C), 115.0, 115.0, 113.2, 86.7, 86.7, 86.2, 84.3, 82.8, 75.1, 72.7, 72.6, 69.9, 68.2, 68.1, 63.0, 55.2 (2C), 34.1, 20.6, 20.4. HRMS (ESI+): m/z calcd for C₆₂H₆₁N₁₀O₁₅S [M + H]⁺: 1217.4033, found: 1217.4052.

### S-(5'-O-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-2'-O-methyl-N<sup>6</sup>-phenoxyacetyladenosyl)-5'-thio-N<sup>6</sup>-phenoxyacetyl-2',3'-O-acetyladenosine 1

To a solution of 7 (890 mg, 0.73 mmol, 1.00 eq) in anhydrous CH₂Cl₂ (5.2 mL) previously passed through an alumina column were added DIEA (0.2 mL, 0.88 mmol, 1.20 eq) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.18 mL, 1.02 mmol, 1.40 eq). The mixture was stirred for 1.5 h at room temperature under argon. After reaction completion, ethyl acetate previously washed with a
saturated aqueous NaHCO₃ was added and the reaction mixture was poured into saturated NaCl/NaHCO₃ 1/1 v/v). The aqueous layer was extracted with ethyl acetate and organic layers were dried over Na₂SO₄. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel chromatography (80-100% DCM in cyclohexane, 1% pyridine then 0-10% acetone in DCM, 1% pyridine) to give a mixture of 50% of the dinucleoside **1** and 50% of the oxidized dinucleoside **8** as a white foam (880 mg). R_f = 0.83 (Cyclohexane/DCM 30:70). ³¹P-NMR (121 MHz, CD₃CN) δ 150.0, 149.8 HRMS (ESI+): m/z calcd for C₇₁H₇₈N₁₂O₁₆PS [M + H]⁺: 1417.5112, found: 1417.5106.

### 4.2.2. Synthesis of dinucleoside phosphoramidite XNs **2**

#### 2',3'-O-acetyl-5'-O-tosyladenosine **10**

To a solution of 5'-O-tosyladenosine **11** (1.0 g, 2.37 mmol, 1.00 eq) in anhydrous pyridine (9 mL) was added acetic anhydride (630 µL, 6.64 mmol, 2.80 eq). The reaction mixture was stirred at room temperature for 18 h under argon. Then, absolute EtOH (9 mL) was added and solvents were removed under vacuum. After coevaporations with toluene (3 x 20 mL), **10** (958 mg, 1.90 mmol, 80%) was obtained as a white solid. R_f = 0.87 (MeOH/DCM 10:90). ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.06 (s, 1H), 7.71 – 7.63 (m, 2H), 7.39 (br s, 2H), 7.34 – 7.25 (m, 2H), 6.15 (d, *J* = 5.3 Hz, 1H), 5.94 (t, *J* = 5.6 Hz, 1H), 5.57 (dd, *J* = 6.0, 4.5 Hz, 1H), 4.51 – 4.27 (m, 3H), 2.36 (s, 3H), 2.09 (s, 3H), 2.01 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.3, 169.1, 156.1, 152.7, 148.8, 145.0, 139.8, 131.7, 129.8, 127.5, 119.2, 85.6, 79.2, 71.8, 69.7, 69.2, 21.1, 20.3, 20.1. HRMS (ESI+): m/z calcd for C₂₁H₂₄N₅O₈S [M + H]⁺: 506.1340, found: 506.1365.

#### 3',5'-O-di-tert-butyldimethylsilyl-2'-O-ethylamino-N-(4-nitrobenzene-sulfonyl)-adenosine **12**

To a solution of **9** (1.42 g, 2.06 mmol, 1.00 eq) in anhydrous DMF (15 mL) were successively added 4-nitrobenzenesulfonamide (831 mg, 4.12 mmol, 2.00 eq), KI (102 mg, 0.62 mmol, 0.30 eq) and K₂CO₃ (851 mg, 6.17 mmol, 3.00 eq). After stirring at 50 °C for 24 h under argon, the reaction was diluted in AcOEt and water. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-2% MeOH in DCM) to give **12** as a pale brown solid (986 mg, 1.55 mmol, 75%). R_f = 0.67 (MeOH/DCM 5:95). ¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 8.37 – 8.26 (m, 2H), 8.16 (s, 1H), 8.09 – 7.95 (m, 2H), 6.65 (t, *J* = 5.6 Hz, 1H), 6.01 (d, *J* = 3.7 Hz, 1H), 5.75 (br s, 2H), 4.41 (t, *J* = 5.0 Hz, 1H), 4.17 (dd, *J* = 4.8, 3.7 Hz, 1H), 4.05 (dt, *J* = 5.3, 2.7 Hz, 1H), 3.97 (dd, *J* = 11.6, 3.1 Hz, 1H), 3.83 (ddd, *J* = 10.1, 5.0, 3.5 Hz, 1H), 3.75 (dd, *J* = 11.6, 2.4 Hz, 1H), 3.61 (ddd, *J* = 10.3, 7.6, 3.4 Hz, 1H), 3.37 – 3.11 (m, 2H), 0.91 (s, 9H), 0.88 (s, 9H), 0.14 – 0.01 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 155.6, 153.5, 150.1, 149.2, 146.3, 138.8, 128.4, 124.5, 120.4, 88.0, 85.3, 83.9, 70.1, 69.7, 61.7, 43.6, 26.1, 25.8, 18.6, 18.1, -4.5, -4.6, -5.2, -5.3. HRMS (ESI+): m/z calcd for C₃₀H₅₀N₇O₈SSi₂ [M + H]⁺: 724.2972, found: 724.2980.

#### N-((3',5'-O-di-tert-butyldimethylsilyl)-2'-O-ethyladenosine)-N-4-nitrobenzenesulfonyl)-5'-deoxy-5'-amino-2',3'-O-acetyladenosine **13**

To a solution of **12** (986 mg, 1.36 mmol, 1.00 eq) in anhydrous DMF (19 mL) were successively added KI (68 mg, 0.41 mmol, 0.30 eq) and K₂CO₃ (564 mg, 4.09 mmol, 3.00 eq). The reaction mixture was stirred at 55°C under argon. Then, **10** (826 mg, 1.64 mmol, 1.20 eq) was added in small portions over 8 h. After stirring at 55°C for additional 16 h under argon, the reaction was diluted in AcOEt (40 mL). Then, a 10% aqueous citric acid solution was slowly added (40 mL) and the aqueous layer was extracted with AcOEt. The combined organic extracts were washed with a saturated aqueous NaHCO₃, with
brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-5% MeOH in DCM) to give **13** as a pale brown foam (806 mg, 0.76 mmol, 56%). R_f = 0.40 (MeOH/DCM 5:95). ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 8.11 – 8.07 (m, 2H), 7.85 – 7.80 (m, 2H), 7.77 (s, 1H), 5.98 (d, J = 3.5 Hz, 1H), 5.95 – 5.77 (m, 6H), 5.64 (t, J = 5.6 Hz, 1H), 4.55 – 4.37 (m, 2H), 4.18 (dd, J = 4.6, 3.6 Hz, 1H), 4.01 – 3.91 (m, 2H), 3.86 – 3.66 (m, 5H), 3.49 (t, J = 5.5 Hz, 2H), 2.13 (s, 3H), 2.08 (s, 3H), 0.91 (s, 9H), 0.87 (s, 9H), 0.15 – 0.01 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 169.7, 169.5, 155.8, 155.6, 153.4, 153.2, 149.9, 149.7, 149.4, 145.5, 139.7, 139.3, 128.5, 124.0, 120.5, 120.2, 87.4, 87.0, 84.8, 83.0, 80.9, 73.1, 71.7, 69.8, 69.5, 61.7, 50.6, 48.3, 26.2, 25.8, 20.7, 20.6, 18.6, 18.2, -4.5, -4.7, -5.2, -5.3. HRMS (ESI+): m/z calcd for C₄₄H₆₅N₁₂O₁₃SSi₂ [M + H]⁺: 1057.4053, found: 1057.4055.

### N-((3',5'-O-di-tert-butyldimethylsilyl)-2'-O-ethyl-N⁶-phenoxyacetyladenosine)-N-4-nitrobenzenesulfonyl)-5'-deoxy-5'-amino-2',3'-O-acetyl-N⁶-phenoxyacetyladenosine **14**

To a solution of **13** (806 mg, 0.76 mmol, 1.00 eq) in anhydrous THF (7.6 mL) were added pyridine (250 µL, 4.57 mmol, 4.00 eq) and Pac₂O (1.31 g, 3.05 mmol, 6.00 eq). After stirring at room temperature for 24 h under argon, the reaction was diluted in AcOEt (40 mL) and washed with saturated aqueous NaHCO₃ (40 mL). The aqueous layer was extracted with AcOEt. The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-5% MeOH in DCM) to give **14** as a white foam (770 mg, 0.58 mmol, 76%). R_f = 0.65 (MeOH/DCM 5:95). ¹H NMR (400 MHz, CDCl₃) δ 9.55 – 9.39 (m, 2H), 8.76 (s, 1H), 8.74 (s, 1H), 8.37 (s, 1H), 8.19 – 8.12 (m, 2H), 8.09 (s, 1H), 7.92 – 7.84 (m, 2H), 7.39 – 7.32 (m, 4H), 7.09 – 7.01 (m, 6H), 6.04 (d, J = 3.2 Hz, 1H), 5.99 (d, J = 4.8 Hz, 1H), 5.90 (dd, J = 5.9, 4.8 Hz, 1H), 5.59 (t, J = 5.6 Hz, 1H), 4.86 (s, 4H), 4.53 – 4.39 (m, 2H), 4.19 (dd, J = 4.6, 3.3 Hz, 1H), 4.03 – 3.92 (m, 2H), 3.91 – 3.65 (m, 5H), 3.58 – 3.37 (m, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 0.90 (s, 9H), 0.86 (s, 9H), 0.15 – 0.02 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 169.7, 169.5, 166.8, 166.7, 157.2, 157.1, 152.9, 152.7, 151.4, 151.3, 150.0, 148.9, 148.5, 145.5, 142.6, 142.1, 130.0, 128.5, 124.2, 123.5, 123.2, 122.6, 115.1, 87.5, 87.0, 84.9, 83.0, 81.1, 72.9, 71.6, 69.6, 68.2 (2C), 61.5, 50.5, 48.4, 26.2, 25.8, 20.7, 20.5, 18.6, 18.1, -4.5, -4.7, -5.3 (2C). HRMS (ESI+): m/z calcd for C₆₀H₇₇N₁₂O₁₇SSi₂ [M + H]⁺: 1325.4783, found: 1325.4797.

### N-(2'-O-ethyl-N⁶-phenoxyacetyladenosine)-N-4-nitrobenzenesulfonyl)-5'-deoxy-5'-amino-2',3'-O-acetyl-N⁶-phenoxyacetyladenosine **15**

To a solution of **14** (770 mg, 0.58 mmol, 1.00 eq) in anhydrous THF (5.8 mL) was added Et₃N·3HF (570 µL, 3.49 mmol, 6.00 eq). The reaction mixture was stirred at 40°C for 24 h under argon. After reaction completion, TEAAc buffer 50 mM, pH 7 was added (5 mL). The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-3% MeOH in DCM) to give **15** as a white foam (451 mg, 0.41 mmol, 71%). R_f = 0.41 (MeOH/DCM 5:95). ¹H NMR (400 MHz, CDCl₃) δ 9.75 – 9.42 (m, 2H), 8.77 (s, 1H), 8.71 (s, 1H), 8.29 – 8.13 (m, 4H), 7.90 – 7.79 (m, 2H), 7.37 – 7.29 (m, 4H), 7.09 – 6.98 (m, 6H), 6.12 (d, J = 5.0 Hz, 1H), 6.00 (t, J = 5.5 Hz, 1H), 5.92 (d, J = 7.0 Hz, 1H), 5.63 (t, J = 5.5 Hz, 1H), 4.88 (s, 4H), 4.74 (dd, J = 7.1, 4.6 Hz, 1H), 4.42 (dd, J = 4.6, 1.3 Hz, 1H), 4.35 (ddd, J = 8.5, 5.2, 3.2 Hz, 1H), 4.30 – 4.25 (m, 1H), 3.95 (dd, J = 13.1, 1.9 Hz, 1H), 3.84 – 3.73 (m, 2H), 3.68 – 3.59 (m, 1H), 3.55 – 3.47 (m, 2H), 3.47 – 3.38 (m, 1H), 3.16 – 3.05 (m, 1H), 2.18 (s, 3H), 2.10 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.9, 169.6, 167.1, 166.9, 157.0 (2C), 152.7, 152.0, 151.2, 150.6, 150.2, 149.1, 148.8, 143.9, 143.7, 143.2, 129.9, 128.5, 124.3, 124.1, 123.5, 122.5, 115.0,
89.2, 88.0, 87.5, 82.1, 81.8, 72.4, 71.4, 70.6, 68.6, 68.2 (2C), 62.9, 51.5, 49.5, 20.6, 20.4. HRMS (ESI+): m/z calcd for C₄₈H₄₉N₁₂O₁₇S [M + H]⁺: 1097.3054, found: 1097.3027.

### N-(4,4'-O-dimethoxytrityl-2'-O-ethyl-N⁶-phenoxyacetyl-adenosine)-N-4-nitrobenzenesulfonyl)-5'-deoxy-5'-amino-2',3'-O-acetyl-N⁶-phenoxyacetyladenosine 16

After three coevaporations of **15** with anhydrous pyridine (30 mL), a solution of **15** (441 mg, 0.40 mmol, 1.00 eq) in anhydrous pyridine (3.1 mL) was heated at 60°C under argon. Then, 4,4'-dimethoxytrityl chloride (204 mg, 0.60 mmol, 1.50 eq) was added in small portions over 30 min. The mixture was stirred for 4 h at 60°C. After cooling to room temperature, a saturated aqueous NaHCO₃ was added. The aqueous layer was extracted with DCM and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0-2% MeOH in DCM, 1% pyridine) to give **16** as a white foam (497 mg, 0.36 mmol, 88%). R_f = 0.67 (MeOH/DCM 5:95). ¹H NMR (600 MHz, CDCl₃) δ 9.55 – 9.41 (m, 2H), 8.71 (s, 1H), 8.68 (s, 1H), 8.62 (m, 3H), 8.26 (s, 1H), 8.22 – 8.18 (m, 2H), 8.06 (s, 1H), 7.93 – 7.87 (m, 2H), 7.77 – 7.57 (m, 1H), 7.47 – 7.39 (m, 2H), 7.39 – 7.24 (m, 6H), 7.24 – 7.18 (m, 1H), 7.07 – 6.99 (m, 6H), 6.86 – 6.79 (m, 4H), 6.07 (d, *J* = 3.5 Hz, 1H), 6.03 (d, *J* = 4.9 Hz, 1H), 5.98 (t, *J* = 5.4 Hz, 1H), 5.61 (t, *J* = 5.4 Hz, 1H), 4.92 – 4.77 (m, 4H), 4.51 – 4.39 (m, 3H), 4.18 (dt, *J* = 5.6, 3.4 Hz, 1H), 3.94 – 3.84 (m, 2H), 3.77 (m, 7H), 3.63 (dd, *J* = 15.3, 8.6 Hz, 1H), 3.60 – 3.48 (m, 2H), 3.42 (dd, *J* = 10.8, 4.1 Hz, 1H), 3.35 (ddd, *J* = 15.1, 6.4, 4.2 Hz, 1H), 2.14 (s, 3H), 2.05 (s, 3H). ¹³C NMR (150 MHz, CDCl₃) 169.9, 169.5, 166.9, 166.8, 158.8, 157.2 (2C), 152.8, 152.7, 151.4, 151.2, 150.0, 150.0, 148.9, 148.5, 144.6 (2C), 142.9, 142.0, 136.1, 135.7 (2C), 130.2, 130.0, 128.7, 128.3, 128.1, 127.1, 124.4, 123.9, 123.6, 123.4, 122.6 (2C), 115.1 (2C), 113.4, 87.7, 86.9, 84.1, 82.5, 81.9, 72.5, 71.7, 69.9, 68.7, 68.2 (2C), 62.9, 55.4 (2C), 50.9, 49.0, 20.7, 20.5. HRMS (ESI+): m/z calcd for C₆₉H₆₇N₁₂O₁₉S [M + H]⁺: 1399.4366, found: 1399.4338.

### N-(5'-O-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-2'-O-ethyl-N6-phenoxyacetyladenosine)-4-nitrobenzenesulfonyl)-5'-deoxy-5'-amino-2',3'-O-acetyl-N6-phenoxyacetyl-adenosine 2

To a solution of **16** (497 mg, 0.36 mmol, 1.00 eq) in anhydrous CH₂Cl₂ (2.5 mL) previously passed through an alumina column were added DIEA (90 µL, 0.50 mmol, 1.40 eq) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (100 µL, 0.43 mmol, 1.20 eq). The mixture was stirred for 1.5 h at room temperature under argon. After reaction completion, ethyl acetate previously washed with a saturated aqueous NaHCO₃ was added and the reaction mixture was poured into saturated NaCl/NaHCO₃ 1/1 v/v). The aqueous layer was extracted with ethyl acetate and organic layers were dried over Na₂SO₄. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel chromatography (50-100% DCM in Cyclohexane, 1% pyridine then 0-5% acetone in DCM, 1% pyridine) to give **2** as a pale brown foam (317 mg, 0.20 mmol, 56%). R_f = 0.63 (Cyclohexane/DCM 50:50). ³¹P-NMR (121 MHz, CD₃CN) δ 149.6, 149.2 HRMS (ESI+): m/z calcd for C₇₈H₈₄N₁₄O₂₀PS [M + H]⁺: 1599.5445, found: 1599.5415.

### 4.2.3. Synthesis of N6-m-chlorobenzyl adenosine 19

### 5'-deoxy-5'-N-azido-2',3'-O-isopropylidene-N6-(3-chlorobenzyl)adenosine 23

To a solution of **22** (500 mg, 1.16 mmol, 1.00 eq) in 1,4-dioxane under argon were added diphenylphosphoryl azide (500 µL, 2.32 mmol, 2.00 eq) and DBU (520 µL, 3.48 mmol, 3.00 eq). After stirring at 25°C for 3 hours, sodium azide (377 mg, 5.80 mmol, 5.00 eq) and 15-crown-5 (23 µL, 0.12 mmol, 0.10 eq) were added and the mixture was stirred at 100°C for 5 hours. After cooling to room temperature, excess of sodium azide was removed by filtration and the filtrate was concentrated to
dryness. The residue was purified by flash column chromatography (silica gel, linear gradient 0 – 2% MeOH in DCM) as the eluent to give **23** (460 mg, 87%) as a white solid. Rf 0.44 (9:1 AcOEt/MeOH). ¹H NMR (600 MHz, CDCl₃) δ 8.40 (s, 1H), 7.81 (s, 1H), 7.42 – 7.32 (m, 1H), 7.26 – 7.23 (m, 3H), 6.33 (t, *J* = 6.1 Hz, 1H), 6.09 (d, *J* = 2.3 Hz, 1H), 5.46 (dd, *J* = 6.3, 2.2 Hz, 1H), 5.07 (dd, *J* = 6.4, 3.4 Hz, 1H), 4.98 – 4.69 (m, 2H), 4.38 (ddd, *J* = 6.5, 5.1, 3.4 Hz, 1H), 3.66 – 3.47 (m, 2H), 1.62 (s, 3H), 1.39 (s, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 154.9, 153.5, 148.6, 140.7, 139.6, 134.7, 130.1, 127.8, 127.8, 125.8, 120.6, 114.9, 90.8, 85.9, 84.3, 82.2, 52.5, 43.9, 27.3, 25.5. HRMS (ESI+): m/z calcd for C₂₀H₂₂ClN₈O₃ [M+H]⁺: 457.1498, Found 457.1505.

**5'-deoxy-5'-N-azido-N6-(3-chlorobenzyl)adenosine 19**

**23** (278 mg, 0.61 mmol, 1.00 eq) was treated at room temperature with TFA in water (8/2, conc. 0.05 M). After stirring for 3 hours, solvents were removed. The residue was diluted with AcOEt (30 mL) and washed with saturated aqueous NaHCO₃ (30 mL). The aqueous layer was extracted with AcOEt (3 x 30 mL) and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0 – 5% MeOH in AcOEt) to give **19** (250 mg, 99%) as a white solid. Rf 0.60 (9:1 AcOEt/MeOH). ¹H NMR (600 MHz, DMSO-d₆) δ 8.46 (br. s, 1H), 8.40 (s, 1H), 8.24 (s, 1H), 7.49 – 7.15 (m, 4H), 5.95 (d, *J* = 5.3 Hz, 1H), 5.57 (d, *J* = 5.8 Hz, 1H), 5.37 (d, *J* = 5.1 Hz, 1H), 4.85 – 4.61 (m, 2H, H₂'), 4.21 (q, *J* = 4.8 Hz, 1H), 4.09 – 3.99 (m, 1H), 3.68 (dd, *J* = 13.2, 7.0 Hz, 1H), 3.56 (dd, *J* = 13.1, 3.7 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 154.8, 153.1, 149.3, 143.2, 140.6, 133.4, 130.6, 127.4, 127.1, 126.3, 120.1, 88.3, 83.4, 78.2, 71.4, 52.2, 42.9. HRMS (ESI+): m/z calcd for C₁₇H₁₈ClN₈O₃ [M+H]⁺: 417.1190, Found 417.1205.

**4.3. Oligonucleotide (ORN-X) syntheses**

Oligonucleotides syntheses were performed on LCAA-CPG solid support (Biosearch Technologies) at 1 µmole scale using an automated DNA synthesizer (Applied Biosystems 394) with TWIST™ synthesis columns (Glen Research) and oligonucleotide synthesis reagents from Biosearch Technologies. 2'-O-PrOM and 2'-O-PivOM ribonucleosides, 2'-O-propargyl adenosine phosphoramidites were purchased from ChemGenes (MA, USA). 3'-cyanine 5 CPG was obtained from Biosearch Technologies.
After ORN elongation, different treatments depending of 5'-extremity (OH, Gppp, ⁷mGppp) of ORN-X conjugates were applied.

For 5'-OH ORN-XS **24-27**, ORN-XN **28-30**, the solid support was treated with a solution of 1M DBU/CH₃CN (2 mL) for 3 min at room temperature. Then, ORN were cleaved from solid support using a 30% aqueous ammonia solution for 3 h at 40°C. The solvents were evaporated under vacuum (in the presence of 500 µL of isopropylamine for ORN sequences containing guanosine) [50]. ORN-X were analyzed and purified by IEX-HPLC, and were characterized by MALDI-TOF MS. ORN-X were desalted using a C₁₈ cartridge Sep-Pak® Classic.

For GpppORN-XS **35-38**, GpppORN-XN **39**, ⁷mGpppORN-XS **45-50**, ⁷mGpppORN-XNs **51-52**, the 5'-capping steps with GDP or ⁷mGDP were performed as described in a previous publication [51]. The deprotection treatment was applied as described for 5'-OH ORN-X using 30% aqueous ammonia solution for GpppORN-X or 7M methanolic ammonia solution for ⁷mGpppORN-X.

For the synthesis of ORN-XTz **31-34**, **40-44**, **53-57** the CPG beads (1 µmole) were poured in a sealed microwaves tube and azide **18** (100 mM solution in 1,4-dioxane, 30 µL, 3.00 eq), CuSO₄ (40 mM solution in water, 10 µL, 0.40 eq), sodium ascorbate (100 mM solution in water, 20 µL, 2.00 eq) and water (20 µL) (water used for dilution was previously flushed under argon for 5 min) were added under argon.
The reaction mixture was heated at 65°C under microwaves for 1 h (400 rpm). Then, solid support was
washed with 1,4-dioxane, a 0.1 M aqueous solution of EDTA, water, MeOH, CH₂Cl₂ and dried under argon. Solid support was treated with a solution of 1M DBU/CH₃CN (2 mL) for 3 min at room temperature and ORN were cleaved from solid support and fully deprotected using a 30% aqueous ammonia solution or a 7M methanolic ammonia solution for 3 h at 40°C. Solvents were evaporated under vacuum (in the presence of 500 µL of isopropylamine for ORN containing guanosine). ORN-X<sup>Tz</sup> were analyzed and purified by preparative IEX-HPLC, and were characterized by MALDI-TOF-MS. ORN-X<sup>Tz</sup> were desalted using a C₁₈ cartridge Sep-Pak® Classic, lyophilized and stored at -20°C.

### 4.4. MTase filter-binding assay (FBA)

The transfer of tritiated methyl from [3H]SAM onto RNA substrate was monitored by filter-binding assay, performed according to the method described previously [62]. The recombinant methyltransferase domain of the non-structural protein NS5 from DV serotype 2 (NS5MTase<sub>DV2</sub>) corresponding to residues 1–296 of NS5 was expressed and purified as described previously [63]. FBA was carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 µM SAM and 0.1 µM 3H-SAM (Perkin Elmer)] in the presence of 0.7 µM synthetic 7mGpppAC₄ and NS5MTaseDV₂ protein (500 nM). The enzyme was first mixed with the compound suspended in water before the addition of RNA substrate and SAM and then incubated at 30°C. Reactions mixtures were stopped after 30 min by their 10-fold dilution in ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filtermat (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol before drying. They were soaked with scintillation fluid (Perkin Elmer), and 3H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux Liquid Scintillation Counter (Perkin Elmer). For IC₅₀ measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y=100/(1+((X/IC50)^(Hillslope))). IC₅₀ is defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity.

### 4.5. Fluorescence polarization (FP)

Compounds containing the sequence that corresponds to the 5' end of dengue virus (DENV) mRNA and 3'-functionalized with cyanine 5 (Cy5) were incubated (at room temperature for 5 min) with increasing concentrations of NS5MTase<sub>DV2</sub> protein in reaction buffer [Tris-HCl 50 mM (pH 8), NaCl 50 mM, DTT 1 mM]. FP measurements were performed in 384-well black plate using a microplate reader (PHERAstar FS; BMG Labtech) with an optical module equipped with polarizer and using excitation and emission wavelengths of 590 and 675 nm, respectively. Dissociation constants (K<sub>d</sub>) were determined using one site specific binding with Hill slope curve fitting with the following equation: Y=Bmax*X<sup>h</sup>/(K<sub>d</sub>*X<sup>h</sup>) (Prism GraphPad software). K<sub>d</sub> is defined as the ligand concentration that binds to half the receptor sites at equilibrium.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Funding

This work was supported by the Agence Nationale pour la Recherche (ANR) under MetInCoV grant agreement n°ANR-21-CO14-0004-01 and Virage project ANR-20-CE11-0024-02, the European Union's Horizon 2020 Research and Innovation program under grants no. 101005077 (the CARE project) and the Fondation pour la Recherche Médicale [FDT202204014965]. R. A-B. thanks University of Montpellier for financial support of his PhD work and ANR for funding the post-doc fellowship. J.T salaries were supported by ANR MetInCoV.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at :

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