The development of nucleic acid therapy requires efficient chemical reactions to connect oligonucleotides with functional chemical modules to improve their stability, promote cellular uptake and achieve specific targeting. In the currently used siRNA therapy, the 3' end of the oligonucleotide is mainly modified with triple-N-acetylgalactosamine (GalNAc)3. However, the implementation of this modification method requires cumbersome multi-step Synthesis and subsequent loading onto a solid phase carrier.
The team of Thomas Carell from the University of Munich published an article entitled "Efficient Tandem Copper-Catalyzed Click Synthesis of Multisugar-Modified Oligonucleotides" on April 12, 2024. They developed a bifunctional linker molecule that can realize multiple sugar click reactions and achieve sugar modification of Oligonucleotides. This modification method has high reaction efficiency and can be completed in a one-pot method.
Fig. 1 Sugar modification of siRNA. (Tölke, et al., 2024)
The siRNA drug Inclisiran is modified with (GalNAc)3 at the 3' end of the sense strand. Since this modification method is expensive both in terms of synthesis and Purification, the authors developed a simple dendrimeric, four-armed tetra-azide linker, which can be used to achieve sugar modification of oligonucleotides in a one-pot method. Using alkyne-modified phosphoramidite monomers, alkyne modifications can be introduced at any position of the oligonucleotide with high yield through standard Solid-phase Synthesis reactions, and then the linker molecule is used for a two-step click reaction, which can directly, quickly and efficiently synthesize sugar-modified oligonucleotides.
The design idea of linker molecule 3 is: one end contains a picolyl-azide and the other end contains three alkyl azides; the reaction rate of the former is much higher than that of the latter, so the authors hope to use this reaction rate difference to perform two-step selective click reactions in succession. First, the authors synthesized linker molecule 3. The starting material triethylenglycol (TEG)-chloride was first converted into the tosyl-TEG-azide, and then reacted with methyl-3,4,5-trihydroxybenzoic acid methyl ester to generate triple-azide-TEG-dendrimer, and then the building block A was obtained by saponification reaction. Meanwhile, 2,5-dicarboxypyridine methyl ester was reduced with NaBH4 to generate picolyl-alcohol, which was then converted to picolyl-azide. Then, it was saponified with LiOH to obtain carboxylic acid. Carboxylic acid was reacted with tert-butyl-(2-(2-(2-aminoethoxy)ethoxy)ethyl) carbamate to obtain PEG-azide-pyridine. Then, PEG-azide-pyridine was Boc-deprotected to obtain compound B, and finally, compound B was coupled with A to generate linker 3.
Fig. 2 Synthesis of linker molecule 3. (Tölke, et al., 2024)
The authors then reacted the alkyne-modified oligonucleotide with linker 3 at room temperature for 30 minutes under shaking. The reaction mixture was then analyzed by HPLC and MALDI-TOF mass spectrometry, which showed complete conversion of the oligonucleotide. Next, a new CuSO4 solution containing THPTA ligand, ascorbate and the corresponding sugar-alkynes was added to the reaction mixture without purification and treatment. A second click reaction was performed using alkyne-bearing glucose (Glc) as an example. The products of the second click reaction were then analyzed by HPLC and MALDI-TOF. MALDI-TOF analysis showed no unreacted starting material, and a major signal peak was also shown in the corresponding HPLC chromatogram. In the entire two-step click reaction, the first click reaction required a reaction time of 10 minutes, and the second click reaction took 1 hour.
Finally, the authors explored whether this two-step Click Reaction could be used to synthesize the oligonucleotide ORN 3 sense 3xGalNAc, which has the same sequence and chemical modification pattern as inclisiran. To this end, the authors prepared the corresponding oligonucleotide ORN 3 sense, which incorporated an octadiynyl-2'-methoxyuridine-phosphoramidite at the 3' end. The oligonucleotide was then subjected to a two-step click reaction with the linker molecule 3 and GalNAc. Subsequently, the reaction mixture was analyzed by HPLC and MALDI-TOF mass spectrometry, and the results showed that the first click reaction was completely converted after 30 minutes, and the second click reaction was completely converted after 1 hour, with a yield of 70-80%.
In this study, Thomas Carell and his team successfully developed a two-step click synthesis method that can quickly convert siRNA and other oligonucleotides into highly modified structures in one pot. Compared with the currently used multi-step solid-phase synthesis, this technology simplifies the process and provides a more economical method for the development of nucleic acid drugs, laying the foundation for the future development of nucleic acid therapeutics.
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