Versatile Site-Specific Conjugation of Small Molecules to siRNA Using Click Chemistry
| Autor: | Md. Rowshon Alam, Haripriya Addepalli, Shigeo Matsuda, Kallanthottathil G. Rajeev, K. Charisse, Chang Geng Peng, Mitsuo Sekine, Takeshi Yamada, K. Mills, K. N. Jayaprakash, Martin Maier, Muthiah Manoharan |
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| Rok vydání: | 2011 |
| Předmět: |
Azides
Molecular Structure Chemistry Stereochemistry Oligonucleotide Organic Chemistry Nucleosides Stereoisomerism Oligonucleotide synthesis Small molecule Catalysis Cycloaddition chemistry.chemical_compound Cyclization Alkynes Click chemistry Moiety Click Chemistry Azide RNA Small Interfering Copper Conjugate |
| Zdroj: | The Journal of Organic Chemistry. 76:1198-1211 |
| ISSN: | 1520-6904 0022-3263 |
| DOI: | 10.1021/jo101761g |
| Popis: | We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line. |
| Databáze: | OpenAIRE |
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