# Synthesis, duplex-forming properties, and enzymatic resistance of oligonucleotides containing N -acylsulfonamide linkages

Romain Amador, Carine Baraguey, Guillaume Clavé, Michael Smietana

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Romain Amador, Carine Baraguey, Guillaume Clavé, Michael Smietana. Synthesis, duplex-forming properties, and enzymatic resistance of oligonucleotides containing N -acylsulfonamide linkages. Organic & Biomolecular Chemistry, 2025, 23 (42), pp.9576-9581. 10.1039/D5OB01382H. hal-05343259

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
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aReceived 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

# Synthesis, Duplex-forming Properties, and Enzymatic Resistance of Oligonucleotides Containing N-acylsulfonamide Linkages

Romain Amador,ª Carine Baraguey,ª Guillaume Clavé*ª and Michael Smietana*ª

We report the synthesis of (2-(*N*-acetylsulfamoyl)ethyl)carbamate (*NAC*)-linked thymidine dinucleotide via the sulfo-click reaction and its incorporation into DNA oligonucleotides. Thermal denaturation studies indicate modest destabilization at duplex ends (~1°C) and stronger effects in the middle (~7°C), while the *NAC* linkage provides complete resistance to nucleases.

Antisense oligonucleotides (ASOs) represent a promising class of therapeutic agents that leverage the sequence-specific binding to target RNA, enabling precise modulation of gene expression. This approach has positioned ASOs as a powerful tool for the treatment of a variety of genetic disorders, cancers, and viral infections.¹, ² Upon binding to the targeted complementary RNA sequences, ASOs induce the recruitment of RNase-H, which selectively cleaves the RNA strand within the resulting RNA/DNA duplex. Alternatively, ASOs can be designed to modulate pre-mRNA splicing through exon skipping, a strategy that alters transcript processing by promoting the exclusion of specific exons. Eighteen ASO drugs are currently approved and more than 170 are in clinical evaluation.³, ⁴ To enable the modulation of critical pharmacokinetic parameters, including satisfactory cell penetration and resistance to nucleases present in biological media, while still maintaining good water solubility, chemical modification of nucleic acids is a prerequisite before their use as therapeutics.⁵ The internucleoside linkage, the

(deoxy)ribose, and the nucleobase have all been altered by a vast variety of modifications to enhance their properties.², ⁴⁻¹⁰ Among those, the internucleoside linkage is of particular importance, notably to enhance the resistance toward enzymatic degradation.⁶ A number of modified internucleoside linkages have been developed, including phosphorothioates, which are used in many therapeutic oligonucleotides currently on the market. The undesirable effects of these modifications, particularly hepatotoxicity,⁷ have prompted researchers to develop new alternatives including totally modified linkages.⁸⁻¹⁸

While most modifications maintain the 5 covalent bonds found in the natural phosphate (PO) linkage (Fig. 1A), several studies have investigated internucleoside linkages comprising 4 to 7 covalent bonds (Fig. 1). Two main types of modifications have been considered: the first retains the phosphate group,¹⁹⁻²¹ such as the 3'-deoxy-3'-(*C*-(hydroxymethyl)) and 5'-deoxy-5'-(*C*-(hydroxymethyl)) (DHM) linkages, which are characterized by an additional bridging methylene at the 3' or 5' side of the modified internucleoside linkage (Fig. 1B).²⁰, ²¹ The second type entirely replaces the natural PO linkage with a different chemical structure. Among these modifications, the formation of a triazole-based internucleoside linkage, efficiently synthesized using the CuAAC click reaction,²², ²³ has proven to be tolerated by polymerases in primer extension reactions (Fig. 1B).¹³, ²⁴⁻²⁸ Additionally, nitrogen-containing modifications, such as amine (5 or 6 bonds),²⁹ (thio)amide (5 or 6 bonds)⁸, ³⁰, ³¹ or thioacetamido³², ³³ moieties (6 bonds, Fig. 1B) have also been widely studied.

Modified internucleoside linkages display a range of effects on duplex stability.

<footnote>ªInstitut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, 1919 route de Mende, 34095 Montpellier, France.</footnote>

<footnote>E-mail: guillaume.clave@cnrs.fr, michael.smietana@umontpellier.fr</footnote>

<footnote>Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See</footnote>

<footnote>DOI: 10.1039/x0xx00000x</footnote>
A Natural PO linkage

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

■ PO linkage, 5 bonds

B Selected examples of previously reported modified internucleoside linkages

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

■ 3'- and 5'- DHM linkage, 6 bonds²⁰,²¹

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

■ Triazole linkage, 4, 6, 7 bonds¹¹,¹³ ²⁴,²⁶

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

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

■ Amine linkage, 6 bonds²⁹

<figure><img src="image_7.png" /></figure>

■ Amide linkage, 4 bonds³⁰

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

■ Diamide linkage, 6 bonds⁸

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

■ Thioacetamide linkage, 6 bond³²

C This work

<figure><img src="image_10.png" /></figure>

■ NAC linkage, 9 bonds

**Fig. 1** A Structure of a dimer comprising a natural 5 bonds PO internucleoside linkage. B Selected chemical structure of modified internucleoside linkages previously described. C This work describing the 9-bonds dephosphono NAC internucleoside linkage.

For example, both 3'- and 5'- DHM linkages reduce hybridization efficiency with complementary DNA or RNA strands, with the 3'-DHM variant also offering greater resistance to exonuclease III.²¹,¹²

Among triazole linkages, destabilization depends on both the position within the strand and the number of covalent bonds (linkages with seven bonds are markedly more disruptive than those with six or four).¹¹, ¹³, ²⁴, ²⁶ Similarly, comparisons between amine and amide bonds show that amide linkages are less destabilizing, likely due to favourable hydrogen bonding.²⁹ Thioacetamido linkages strongly destabilize DNA duplexes but slightly stabilize RNA.³² Amide-containing linkages, also explored in PNAS,³⁴ were found to be specifically useful for RNA recognition.³⁵, ³⁶ Finally, while sulfamate linkages mildly destabilize DNA, sulfamate/sulfamide variants tend to enhance duplex stability.

¹⁷, ³⁷ To the best of our knowledge, no studies have described modified internucleoside linkages exceeding 7 covalent bonds.

Recently, the N-acylsulfonamide moiety has emerged in an increasing number of pharmaceutical compounds across diverse therapeutic areas, primarily owing to its favourable physicochemical properties.³⁸⁻⁴¹ Furthermore, this functional group has been successfully employed as a biosostere of carboxylic acids and phosphates, further highlighting its versatility in medicinal chemistry.⁴²⁻⁴⁴ Notably, dinucleotides featuring an N-acylsulfonamide internucleoside linkage, preserving the five covalent bonds characteristic of the natural PO backbone, were previously reported by the groups of Matteucci⁴⁵ and Widlanski.⁴⁶ These constructs were synthesized *via* coupling reactions between 4'-carboxylic acid and 3'-sulfonamide nucleosides, employing DCC as a carboxylic acid activator, and yielded moderate efficiencies of 65% and 66%, respectively. However, the preparation of 3'-sulfonamide
nucleosides proved particularly demanding, with an overall yield below 8% over a seven-step sequence.⁴⁶ The resulting dimer was incorporated into an oligonucleotide by Matteucci et al. using H-phosphonate chemistry and was only evaluated through its hybridization properties against a complementary RNA strand. To date, no phosphoramidite dimer bearing an N-acylsulfonamide linkage has been reported.

Building on our previous work on cyclic dinucleotides featuring the N-acylsulfonamide linkage,⁴⁷ we decided to evaluate a (2-(N-acetylsulfamoyl)ethyl)carbamate (NAC) internucleoside linkage including 9 bonds (Fig. 1C). Thereby, here we describe the highly efficient synthesis of a NAC-linked phosphoramidite thymidine dinucleotide 6, prepared via the sulfo-click reaction as key step. This dimer was successfully incorporated into several ODNs using standard phosphoramidite chemistry. The resulting structures were characterized for their ability to form duplexes with complementary DNA or RNA strands and their resistance against nuclease digestion was evaluated.

Despite comprising a relatively high number of bonds (9), the presence of both carbamate and N-acylsulfonamide moieties in the NAC linkage may play a more critical role in maintaining duplex stability than the overall number of bonds within the backbone. Indeed, previous studies by Stork et al. have demonstrated that a 5 bonds amine internucleoside linkage causes significant duplex destabilization ($ΔT_m$ = -13.1°C), whereas a 6 bonds amide linkage is much less disruptive ($ΔT_m$ = -4.1°C) during the hybridization of modified ODNs with their complementary ssRNA. These findings suggest that the chemical features of the linkage, in addition to bond number, are key determinants of duplex stability, preventing straightforward comparisons based on bond count alone.²⁹

Given the importance of efficient synthetic strategies, we employed the sulfo-click reaction, a robust and high-yielding approach that we have recently applied for various applications in the field of nucleic acid chemistry such as the preparation of 5'-nucleoside bioconjugates,⁴⁸, ⁴⁹ the synthesis of SARS-CoV-2 RNA inhibitors cap N7-guanine-methyltransferase nsp14⁵⁰ or the synthesis of original cyclic dinucleotides.⁴⁷

Foremost, the synthesis of sulfonylazide 3 was carried out in 2 steps starting from 5'-O-DMTr-thymidine 1 (Scheme 1) following protocols previously described on A and G.⁴⁷ The free 3'-hydroxyl was engaged with 4-nitrophenyl chloroformate in the presence of DIEA in an activated carbonate forming reaction leading to compound 2. Thereafter, the carbamate 3 was obtained using 2-aminoethanesulfonyl azide⁴⁸ in basic medium. Then, the sulfo-click reaction between the sulfonyl azide 3 and the thioacid 4 (obtained as previously described in three steps with a 47% overall yield⁴⁸) was performed under aqueous conditions [in a 80:20 NMP/H₂O mixture with the addition of NaHCO₃] to yield dimer 5 (Scheme 1). As expected, the reaction allowed the complete conversion of the reactants within 10 min and resulted in a high isolated yield of 88%. In the last step, phosphitylation of the 3'-hydroxyl group of 5 produced the desired phosphoramidite building block 6. The

modified dimer 6 was obtained with a very good overall yield of about 43% over 4 steps from 5'-O-DMTr-thymidine 1 which compares favourably to the literature, owing to the efficiency of the sulfo-click reaction. Noteworthy, all the N-acylsulfonamide derivatives were isolated under the form of deprotonated salts due to their low pKa values.³⁸

We then investigated the influence of the NAC linkage at different positions within a specific 12-mer ODN. First, we incorporated it in the middle of the sequence to accurately assess its impact on duplex stability with complementary strands. Next, we placed the NAC linkage at the extremities to evaluate its resistance to exonuclease digestion. The synthesis of NAC-linked ODNs was performed on a 1-micromolar scale using an automated DNA synthesizer. After optimizing the coupling time for phosphoramidite dimer 6 on our target sequence (ODN2), we established it at 20 minutes (27% overall yield, see ESI Table S1), as the conventional 30 s coupling time led to a very low incorporation rate (<1% overall yield, see ESI Table S1). This reduced efficiency is likely due to the length and flexibility of the NAC linkage, which may cause steric hindrance near the phosphoramidite function. Noteworthy, whereas the synthesis of ODN1, ODN2, and ODN4 using classical dT CPG did not present any significant issues with full cleavage after 12 h at 55°C in 28% aq. $NH_3$, the use of universal CPG solid support with a Q-linker for ODN3, ODN5, and ODN6 required at least 12 h at 85°C in 28% aq. $NH_3$ for complete cleavage. After optimizing the cleavage procedure, satisfactory synthetic yields were obtained. Finally, the modified ODNs 1-6 were successfully isolated and characterized after RP-HPLC purifications.

Next we evaluated the influence of these modified ODNs through thermal denaturation analysis in the presence of complementary DNA or RNA strands (Table 1). We were pleased to observe that the incorporation of the modified NAC thymidine dinucleotide at the 5' (ODN2) or 3' (ODN3) extremities resulted in only a slight destabilization of the duplexes formed ($ΔT_m$ ~ -1°C) with the corresponding complementary DNA strands, thereby paving the way for potential gapmer application.⁵¹ This demonstrates that despite the length of the NAC linkage, its flexibility does not prevent the nucleobases from establishing hydrogen bonds with their counterparts. In contrast, inserting the modified NAC thymidine dinucleotide at positions 6 and 7 of the sequence (ODN4), resulted in a more significant destabilization regardless of the DNA ($ΔT_m$ = -7.3°C) or RNA ($ΔT_m$ = -8.1°C) nature of the complementary strand (Table 1). Additionally, with sequences carrying two (ODN5) or three (ODN6) NAC linkages, $T_m$ values corresponded precisely to the values calculated by summing the individual destabilizations observed in ODN2-4. Given the observed loss of duplex stability induced by the NAC linkage, either at the sequence ends or in the middle, we performed an investigation using NMR spectroscopy on dimer 5', the 5'-OH deprotected derivative of dimer 5 (see ESI S37-S42).
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<figure><img src="image_11.png" /><figcaption>**Scheme 1** Synthesis of NAC-linked phosphoramidite dimer 6. Compound 4 was synthesized in three steps with a 47% overall yield.⁴⁸</figcaption></figure>

<table><caption>**Table 1** T<sub>m</sub> values of the duplexes formed between ODN1–6 and their complementary ssDNA or ssRNA<sup>a</sup></caption><thead><tr><th rowspan="2"></th><th rowspan="2">Sequences<sup>a</sup></th><th colspan="2">Complementary strand<br>T<sub>m</sub> (ΔT<sub>m</sub>)/°C</th></tr><tr><th>DNA</th><th>RNA</th></tr></thead><tbody><tr><td>ODN1</td><td>5'-d(TTCTGTTACATT)-3'</td><td>36.7</td><td>38.2</td></tr><tr><td>ODN2</td><td>5'-d(T<sub>NAC</sub>TCTGTTACATT)-3'</td><td>35.9 (-0.8)</td><td>36.2 (-2)</td></tr><tr><td>ODN3</td><td>5'-d(TTCTGTTACAT<sub>NAC</sub>T)-3'</td><td>35.7 (-1)</td><td>36.1 (-2.1)</td></tr><tr><td>ODN4</td><td>5'-d(TTCTGT<sub>NAC</sub>TACATT)-3'</td><td>29.4 (-7.3)</td><td>30 (-8.1)</td></tr><tr><td>ODN5</td><td>5'-d(T<sub>NAC</sub>TCTGTTACAT<sub>NAC</sub>T)-3'</td><td>34.9 (-1.8)</td><td>33.9 (-4.3)</td></tr><tr><td>ODN6</td><td>5'-d(T<sub>NAC</sub>TCTGT<sub>NAC</sub>TACAT<sub>NAC</sub>T)-3'</td><td>27.3 (-9.4)</td><td>-</td></tr></tbody></table>

<sup>a</sup> Conditions: 10 mM sodium cacodylate buffer (pH 7), 100 mM NaCl, and 3 µM of each ODN. The T<sub>m</sub> values reflect the average of three measurements. Target DNA sequence: 5'-AATGTAACAGAA-3'. Target RNA sequence: 5'-AAUGUAACAGAA-3'. All linkages except for NAC are phosphodiesters.

These studies show that, despite the substitution of the PO linkage by the NAC one, riboses retain a preference for a South-type conformation, with nucleobases orientated *anti*, as observed in the native dimer dTpdT. However, evolution of coupling constants as a function of temperature indicates that the conformation of the ribose bearing the *N*-acylsulfonamide moiety at its C4' position (T1) is more rigid than that of the conventional 5'-OH ribose (Table S8, page S38).

Numerous structural studies have suggested a strong correlation between the rotation of the C4'-C5' bond, the sugar pucker, and the nucleobase position. In unmodified nucleosides and nucleotides, the C4'-C5' bond typically adopts a *gauche-gauche* (+sc) conformation. The absence of protons on the 5' carbon due to the replacement of methylene by carbonyl in T1 prevents the determination of the orientation around the C4'-C5' bond from NMR data. Interestingly, in the X-ray structure of 5'-carboxylic acid thymidine, the deoxyribose adopts a C2'-endo envelope (2E) conformation and an *anti*-orientation of the nucleobase relative to the sugar, with the C4'-C5' bond deviating from its usual *gauche-gauche* arrangement.⁵² This deviation in the γ angle results from the C5' sp2 hybridization and can be expected in the NAC dimer, where similarly the co-planarity of C5', C4', O5' and N atoms would systematically bring the N atom in an unfavourable eclipsed conformation when O5' adopts a staggered conformation. The presence of carbonyl in the 5' position on the dimer model 5' leads to a loss of sugar flexibility in the corresponding residue. Although we cannot directly transpose this result at the oligonucleotide level, one can nevertheless assume that the constraint induced by the carbonyl group within the *N*-acylsulfonamide moiety of the NAC linkage could shift the deoxyribose from its usual conformation, interfering with base pairing and stacking.
Next, enzymatic stability studies using both Calf Spleen
phosphodiesterase (CSPDE) and snake venom
phosphodiesterase 1 (SVPDE, from *Crotalus adamanteus*
venom) were conducted as these enzymes are well-
characterized exonucleases that selectively cleave DNA from
the 5' and 3' ends, respectively. Concerning CSPDE (see ESI
pages S24-S31), the NAC modified internucleoside linkage
demonstrated complete resistance on the 5'-modified **ODN2**
over 48 h. (ESI Fig. S6). Regarding SVPDE (see ESI pages S32-
S37), although the NAC linkage slightly slowed the enzymatic
activity (t₁/₂ = 1 min for **ODN1** and 2 min for **ODN3**, ESI figure
S17), as it has already been reported several times in the
literature, the nuclease was able to "jump" over the modified
linkage to hydrolyze the rest of the sequence of the 3'-
modified **ODN3**,⁶ leading to mononucleotides and the intact
5'-MP-**T**<sub>NAC</sub>T dimer.

In summary, we applied the sulfo-click reaction with a 3'-
sulfonylazide and a 4'-thioacide thymidine synthon leading to
the negatively charged **T**<sub>NAC</sub>T dimer 5 that was then converted
into the phosphoramidite derivative 6 with an overall yield of
about 43% over 4 steps. The combined hybridization data
indicate that the incorporation of the NAC linkage within
oligonucleotides leads to substantial destabilization when
positioned centrally within the sequence. In contrast, its
placement at the terminal regions resulted in only a minor
reduction in the stability of duplexes formed with
complementary DNA or RNA strands. Finally, the 9-bond NAC
linkage induced less destabilization of duplex formation than
several previously reported 5-bond modifications. This
improved performance is likely attributable to the greater
conformational flexibility afforded by the extended nine-bond
backbone. These findings open new perspectives for the
development of modified internucleoside linkages with
extended backbones, while maintaining sufficient flexibility to
facilitate nucleobase pairing. Given the interest in the N-
acylsulfonamide linkage for therapeutic molecules, we will
further pursue the development of shorter NAC linkages.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data supporting the findings of this study are provided
within the main text and the Supplementary Information,
including detailed synthetic procedures, oligonucleotide
sequences and synthesis protocols, purification methods, and
characterization data. Characterization comprises NMR, UV-vis
and mass spectrometry analyses, as well as thermal
denaturation (T<sub>m</sub>) and exonuclease assays.

Acknowledgements

The authors thank the Agence Nationale de la Recherche (ANR
"TALAN"-ANR-19-CE07-0004-01) for financial support.

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