Endowing RNase H-Inactive Antisense with Catalytic Activity: 2-5A-Morphants
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- 作者：Longhu Zhou ; Edgar R. Civitello ; Nidhi Gupta ; Robert H. Silverman ; Ross J. Molinaro ; David E. Anderson ; and Paul F. Torrence
- 刊名：Bioconjugate Chemistry
- 出版年：2005
- 出版时间：March 2005
- 年：2005
- 卷：16
- 期：2
- 页码：383 - 390
- 全文大小：140K
- 年卷期：v.16,no.2(March 2005)
- ISSN：1520-4812
文摘
A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2']np5'A)to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5Astarting material, commercially and readily available synthons for solid-phase synthesis were adaptedfor larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'
5')adenylyl(2'5')adenylyl(2'5')adenosine (p5'A2'p5'A2']2p5'A2', tetramer 2-5A, 9) was synthesized starting with2',3'-O-dibenzoyl-N6,N6-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl)adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection,generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protectedtetramer 2-5 A. The title product 9 p5'A(2'p5'A)3 (tetramer 2-5A) was obtained through phosphorylationof the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups usingconcentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 
C and tetrabutylammonium fluoride(TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisensechimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalizedphosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2']2p5'A).The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel2-5A-antisense chimera had significant biological activity, thereby providing another potential toolfor RNA ablation.
5')adenylyl(2'5')adenylyl(2'5')adenosine (p5'A2'p5'A2']2p5'A2', tetramer 2-5A, 9) was synthesized starting with2',3'-O-dibenzoyl-N6,N6-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl)adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection,generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protectedtetramer 2-5 A. The title product 9 p5'A(2'p5'A)3 (tetramer 2-5A) was obtained through phosphorylationof the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups usingconcentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 C and tetrabutylammonium fluoride(TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisensechimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalizedphosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2']2p5'A).The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel2-5A-antisense chimera had significant biological activity, thereby providing another potential toolfor RNA ablation.