Thioacid / Azide Ligation Applied to Nucleic acids as a new tool for chemical biology – TALAN

The implementation of the sulfo-click reaction on nucleic acids requires the development of new methods of derivatization of nucleoside monomers in order to incorporate them within oligonucleotide sequences. To achieve this goal, we aim to 1) carry out the sulfo-click reaction on nucleosides having a thioacid function 2) to extend the applicability of the sulfo-click reaction to the synthesis of original analogues of cyclic dinucleotides (CDN) and the implementation of bioconjugation reactions on modified ODN 3) to implement the sulfo-click reaction on a DNA template 4) to set up a method for the detection of miRNAs using the DNA templated sulfo-click reaction. With this project we aim to provide to the scientific community a new tool for bioconjugation reactions, complementary to the reactions already used. Finally, the implementation of this reaction on a DNA template will allow the development of a new efficient method for the detection of miRNA in biological media.

We first concentrated our efforts on the functionalization at the 4’ position of different nucleosides with the thioacid function in order to be able to study the sulfo-click reaction on modified nucleosides. This has been done on thymidine, cytidine and guanosine in the deoxyribonucleotide series, but also on uracil in the ribonucleotide series. Several molecules of interest have been functionalized with the sulfonyl azide function and the sulfo-click reaction has been implemented. The kinetic of the reaction was studied, demonstrating its interest for bioconjugation applications in aqueous media (k ~ 2.0 × 10-1 M-1s-1 at 25°C).
These results were published in 2020 in the journal Org. Lett. and were noticed in the journal Synfacts in the same year. In addition, we were approached by an editor of the journal Curr. Protoc. Nucleic Acid Chem. to publish the detailed protocols of this work.
Among the different nucleic acid structures known for their medical applications, cyclic dinucleotides (CDN) emerge for the targeting of the STING protein (Stimulator of interferon genes). This protein is a key element in the functioning of the innate immune response by stimulating the production of type I interferon, which will limit the infection of a pathogen in neighboring cells. Several recent studies have shown the great potential of stimulation of the STING protein by synthetic CDN. Thus we began the study the synthesis of original CDN having an internucleoside N-acylsulfonamide linkage and on the other hand a phosphate or phosphorothioate linkage. Despite many difficulties induced by the presence of the N-acylsulfonamide linkage, we recently managed to obtain a first CDN.

Over the next few months, our goal is to move forward and possibly complete the work on new CDN synthesis. In addition, we are considering the synthesis of modified ODN comprising the N-acylsulfonamide linkage. Their physico-chemical properties will be studied (aqueous solubility, resistance to nucleases, stability of the duplexes formed with a complementary ODN) for possible applications in the context of therapeutic nucleic acids. (i.e. antisense ODN, siRNA, aptamers). Thereafter, we plan to study the incorporation of the thioacid and / or the sulfonyl azide functions on ODN in order to synthesize bioconjugates, which will pave the way for the study of DNA templated sulfo-click ligation.

1. Clavé, G.; Dursun, E.; Vasseur, J. J.; Smietana, M., An Entry of the Chemoselective Sulfo-Click Reaction into the Sphere of Nucleic Acids. Org. Lett. 2020, 22, 1914-1918.
Highlited in Synfact, Trauner, D.; Shemet, A., Sulfo-Click Conjugation. Synfacts 2020, 16, 0600.
2. Clavé, G.; Vasseur, J. J.; Smietana, M., The Sulfo-Click Reaction and Dual Labeling of Nucleosides. Curr. Protoc. Nucleic Acid Chem. 2020, 83, e120
3. Clavé, G.; Reverte, M.; Vasseur, J.-J.; Smietana, M., Modified internucleoside linkages for nuclease-resistant oligonucleotides. RSC Chem. Biol. 2021, 2, 94-150.

DNA templated chemical reactions are based on the specific pairing properties of complementary nucleic acid strands. The latter are exploited to force spatial proximity between reactive groups of modified ODN in order to dramatically accelerate a given reaction at very low concentrations. Numerous organic reactions have been studied in this context but despite their superior selectivity and efficiency, the use of bioorthogonal and biocompatible click reactions remains surprisingly low. We believe that the development of the Thioacid/Azide Ligation (TAL) has the potential to provide enhanced templated ligation performances as well as a new tool for DNA bioconjugation. In order to exploit this new process, we plan to develop original nucleoside synthons functionalized with the thioacid and sulfonylazide units. First the TAL will be optimized under conventional synthesis conditions in aqueous buffers on different nucleosides in DNA and RNA series. Once this goal achieved we intend to demonstrate the interest of the TAL applied to nucleic acids as a surrogate click reaction. In addition, a series of cyclic dinucleotide analogs (CDN) crucial for the immune system will also be considered with adenosine and guanosine. Over the past few years CDN have shown significant potential because of their biological activity, particularly in the context of new methods of cancer treatment. The final objective of the TALAN project is to develop a new nucleic acid templated chemical reaction. Significant efforts will be made to incorporate the appropriate synthons on oligonucleotides (ODN) in DNA and RNA series. Then the DNA templated TAL will be optimized by focusing in particular on a version using non-electrodeficient alkyl azides. Indeed a new reaction pathway with lower activation energy barrier (reduction of the entropic factors) will be established due to the high effective molarity obtained with a DNA template. This would allow us to describe for the first time a version of the TAL performed at room temperature in a biocompatible medium using a non-electrodeficient azide. The final goal of the TALAN project is to implement this ligation on RNA templates of biological interest and apply it to microRNAs (miRNAs) in order to develop a new effective method for their detection and their quantification within complex biological media. Indeed miRNAs are small (19 to 24-mer) endogenous molecules that negatively regulate gene expression. RNA based diagnostics has the potential to play a key role in the management and the monitoring of many diseases due to significant variations of their concentration in biological solid (tissues, cells) or liquid (saliva, blood, urine, …) samples. The optimization of the chemical structure of the ODN involved will require many efforts to improve the turnover of this catalyzed reaction and ultimately increase the sensitivity of this method.