Endowing RNase H-inactive antisense with catalytic activity: 2-5A-morphants.
A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-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 protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.
Zhou
Longhu
L
Department of Chemistry, Northern Arizona University, Box 5698, Flagstaff, Arizona 86011-5698, USA.
Civitello
Edgar R
ER
Gupta
Nidhi
N
Silverman
Robert H
RH
Molinaro
Ross J
RJ
Anderson
David E
DE
Torrence
Paul F
PF
D000970:Antineoplastic Agents / Q000138:chemical synthesis; D000998:Antiviral Agents / Q000138:chemical synthesis; D002384:Catalysis; D016373:DNA, Antisense / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology; D004722:Endoribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009025:Morpholines; D060172:Morpholinos; D012367:RNA, Viral / Q000187:drug effects / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D016373:DNA, Antisense; D009025:Morpholines; D060172:Morpholinos; D012367:RNA, Viral; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D016914:Ribonuclease H