| Project/Area Number |
60570972
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Chemical pharmacy
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| Research Institution | Hokkaido University |
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Principal Investigator |
MIURA Kazunobu Hokkaido University, Pharmaceutcal Sciences, Inotructor, 薬学部, 助手 (70001980)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Oligonucleotide synthesis / Phosphite procedure / ダイマー単位を用いたホスファィト法 |
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| Research Abstract |
Improvement of synthetic method of oligonucleotides using automated synthesizer has been performed by using dimer unit as condensation unit in place of monomer unit.
5'-O-Dimethoxytritylthymidine 3'-(o-chlorophenyl)phosphate was synthesized from thymidine by dimethoxytritylation and subsequent phosphorylation using o-chlorophenyl phosphoroditriazolide in a good yield. Preparation of dinucleoside monphosphate was carried out by condensation of unprotected thymidine to dimethoxytritylthymidine 3'-(o-chlorophenyl)phosphate in presence of MSNT as condensing agent. The products of the condensation contained byproduct which had 3'-3' linkage and removed by silica gel column chromatography. Then, major product having 3'-5' linkage was phosphitylated with methyl-N,N-diisopropylphosphoramidite in 85% yield. Thus, the dimer unit for the blockwise synthesis of oligonucleotides was prepared conveniently in good yield.
The synthesis of pentadecathymidylate was performed by automated synthesizer which was attached with thymidine-dimer unit solution. The condensation of dimer was repeated 7 times and oligonucleotide bound in polymer support was cleaved by aqueous ammonia treatment. Then, oligonucleotide synthesized was purified by reversed phase HPLC and susequent anion exchange HPLC. The yield was 39 A260 units (30%) When 1 umol of thymidine-linked polymer support was used. This result is excellent, so that the condensation of dimer unit in place of monomer unit is useful and synthetic method using automated synthesizer should be improved.
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