| Project/Area Number |
16350085
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemistry related to living body
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| Research Institution | The University of Tokyo |
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Principal Investigator |
WADA Takeshi The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor (90240548)
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| Project Period (FY) |
2004 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥16,500,000 (Direct Cost: ¥15,600,000、Indirect Cost: ¥900,000)
Fiscal Year 2007: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2006: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2004: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | RNA synthesis / 2'-O-protecting group / solid-phase synthesis / RNAi / siRNA / nucleic acid drugs / automated synthesis / nucleic acid analogs / ホスホロアミダイト法 / 核酸合成 / アンチセンス核酸 / オリゴヌクレオチド / 大量合成 / 液相合成 / 脱水縮合剤 / DNA合成 / リン原子就職核酸 / フルオラス合成法 / フルオラス溶媒 / 固相合成法 / グリーンケミストリー / ホスファイト / 官能基選択的ホスフィチル化 / 位置選択的ホスフィチル化 / ルイス酸 |
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| Research Abstract |
Chemically synthesized oligoribonucleotides have become increasingly important since the recent discovery of small interfering RNAs (siRNAs), micro RNAs (miRNAs), as well as numerous noncoding RNAs (ncRNAs). The mammalian siRNAs consist of only 21-23 nucleotides, and their gene silencing ability enables us to create, in principle, new nucleic acid-based therapeutic agents for many diseases. In this research project, a novel method for the chemical synthesis of RNA using 1-(2-cyanoethoxy)ethyl (CEE) as a 2'-hydroxy protecting group has been developed. A CEE group was introduced to the 2'-position of N-acyl-3',5'-O-silyl-protected ribonucleosides regioselectively under acidic conditions. The 2'-O-CEE group was found to be stable in an aqueous or ethanolic ammonia and was quickly removed by treatment with anhydrous tetrabutylammonium fluoride (TBAF). In the deprotection reaction of the CEE group with TBAF, nucleobases were alkylated to some extent by acrylonitrile via a Michael-type addit
… More
ion. Several acidic compounds were tested as scavengers of acrylonitrile and nitromethane was found to be highly effective to inhibit the nucleobase alkylation. The solid-phase synthesis of oligoribonucleotides (4, 10 and 21mers) was carried out and the products were obtained in good yields. One advantage of the 2'-O-CEE-protected phosphoramidite approach is the highly efficient synthesis of the monomer units since the CEE group can be introduced regioselectively to the 2'-hydroxy group of N-acyl-3',5'-O-silyl-protected ribonucleosides under acidic conditions. The 2'-O-CEE group is quite stable under the basic conditions prescribed for the deprotection of N-acyl and phosphate protecting groups, and is quickly removed by treatment with TBAF. Thus the concomitant use of the 2'-O-CEE with N-acyl and 2-cyanoethyl phosphate protecting groups enables a reliable two-step deprotection procedure, first with ammonia, then with TBAF. In comparison with the conventional 2'-O-protected phosphoramidites, the less sterically hindered 2'-O-CEE-protected phosphoramidites are more reactive and advantageous for the synthesis of longer oligomers. Less
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