Versatile Site-Specific Conjugation of Small Molecules to siRNA Using Click Chemistry

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• 作者：Takeshi Yamada ; Chang Geng Peng ; Shigeo Matsuda ; Haripriya Addepalli ; K. Narayanannair Jayaprakash ; Md. Rowshon Alam ; Kathy Mills ; Martin A. Maier ; Klaus Charisse ; Mitsuo Sekine ; Muthiah Manoharan ; Kallanthottathil G. Rajeev
• 刊名：Journal of Organic Chemistry
• 出版年：2011
• 出版时间：March 4, 2011
• 年：2011
• 卷：76
• 期：5
• 页码：1198-1211
• 全文大小：1274K
• 年卷期：v.76,no.5(March 4, 2011)
• ISSN：1520-6904

文摘

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鈭抣igand conjugates using the copper-catalyzed azide鈭抋lkyne 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 鈥渃lick conjugation鈥? and the 鈥渃lick conjugation鈥?on an immobilized and completely protected alkyne鈭抩ligonucleotide scaffold. Nucleosides bearing 5鈥?alkyne moieties were used for conjugation to the 5鈥?end of the oligonucleotide. Previously described 2鈥? and 3鈥?O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3鈥?and 5鈥?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鈭抩ligonucleotide 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鈭抩ligonucleotide prior to deprotection was the most efficient 鈥渃lick conjugation鈥?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.