| Project/Area Number |
14208074
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | TOKYO INSTITUTE OF TECHNOLOGY |
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Principal Investigator |
SEKINE Mitsuo TOKYO INSTITUTE OF TECHNOLOGY, FACULTY OF BIOSCIENCE AND BIOTECHNOLOGY, PROFESSOR, 大学院・生命理工学研究科, 教授 (40111679)
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| Co-Investigator(Kenkyū-buntansha) |
SEIO Khoji TOKYO INSTITUTE OF TECHNOLOGY, FRONTIER COLLABORATIVE RESEARCH INSTITUTE, ASSOCIATE PROFESSOR, フロンティア創造共同研究センター, 助教授 (20313356)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥43,940,000 (Direct Cost: ¥33,800,000、Indirect Cost: ¥10,140,000)
Fiscal Year 2003: ¥15,210,000 (Direct Cost: ¥11,700,000、Indirect Cost: ¥3,510,000)
Fiscal Year 2002: ¥28,730,000 (Direct Cost: ¥22,100,000、Indirect Cost: ¥6,630,000)
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| Keywords | DNA CHEMICAL SYNTHESIS / NUCLEOBASE / ACTIVATOR / PROTECTING GROUP / LINKER / SOLID PHASE SYNTHESIS / PHOSPHORAMIDITE METHOD / OLIGODEOXYNUCLEOTIDE / 塩基部位無保護DNA合成法 / プロントンブロック法 / ホスファチル化 / DNAの化学合成 / キャップ化反応 / ン-メチルピロリドン / 溶媒効果 |
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| Research Abstract |
Previously, Ο-selective phosphorylation on polymer supports in the N-unprotected phosphoramdite method could not be carried out since the amino groups of dA and dC have high reactivity toward tervalent phosphorus(III)-type phosphitylating reagents. In this paper, we developed a new coupling strategy named 'activated phosphite method' in which the phosphitylation reaction is mediated by phosphite triester intermediates such as compound 1. In the solution-phase synthesis of TpA and TpC derivatives, the condensation by use of HOBt showed almost 100% Ο-selectivity and, they could be isolated in high yields. Subsequently, we examined the Ο-selectivity of the phosphorylation by use of various 1-hydroxybenzotriazole-and phenol-type promoters in the synthesis of dimers (ApT, CpT, and GpT) and trimers (TpApT, TpCpT, and TpGpT) on polymer supports. Application of HOBt as the promoter to the solid-phase synthesis resulted in excellent Ο-selectivity of more than 99.7%. We explained this Ο-selectiv
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ity based on the FMO interactions between the reactive intermediates and the nucleophiles such as the amino or hydroxyl groups of nucleosides. It was also found that the Ο-selectivity decreased in the condensation by use of HOBt-type promoters with an increase of the acidity of the promoter. The detailed studies on the mechanism of our present approach disclosed that, during this condensation, a phosphite benzotriazolyl ester intermediate is converted via rearrangement into the corresponding pentavalent phosphorus species which was found to be still reactive to hydroxy functions. It was estimated that contribution of this pentavalent species to the overall phosphorylation is at most 4 -10% in the condensation. Furthermore, we succeeded in the synthesis of longer oligonucleotides not only by a manual operation but also by a DNA synthesizer. The utility of our new method was demonstrated by the successful synthesis of an alkali-labile modified oligodeoxyribonucleotide having 4-N-acetyl deoxycytidine residues. Less
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