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. 2012 Aug 28;10(32):6491-7.
doi: 10.1039/c2ob25647a. Epub 2012 Jul 5.

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

José M Ibarra-Soza  1 , Alexi A Morris, Prasanna Jayalath, Hayden Peacock, Wayne E Conrad, Michael B Donald, Mark J Kurth, Peter A Beal
Affiliations

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

José M Ibarra-Soza et al. Org Biomol Chem. .

Abstract

Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

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Figures

Figure 1
Figure 1
Eight purine 7-position modifications in duplex RNA.
Figure 2
Figure 2
Azides synthesized for their use in the copper-catalyzed azide/alkyne cycloaddition with oligonucleotides containing 7-ethynyl-8-aza-7-deazaadenosine.
Figure 3
Figure 3
Thermal denaturation (Tm) of the 12-mer duplexes containing adenosine and the 7-substituted 8-aza-7-deazaadenosine analogues; propargylamine, ethynyl, and triazoles I–VI opposite the four natural bases.
Figure 4
Figure 4
(A) Sequence of siRNA used in this study showing sites of modification of the guide strand and the structures of the modifications tested. All siRNAs were prepared with a 5′-phosphorylated guide strand (p). (B) Knockdown activty of modified siRNAs. Activity is reported as the ratio of Renilla/firefly luciferase signal at 0.03 nM transfected siRNA in HeLa cells. buffer = no added siRNA, unmodified = siRNA with no modifications.
Scheme 1
Scheme 1
Synthesis of 7-propargylamine- (6) and 7-ethynyl- (7) phosphoramidites: (a) N′-(2-propynyl)-2″’,2‴,2″-trifluoroacetamide, Pd(PPh3)4, CuI, Et3N, DMF, rt, 91%. (b) ethynyltrimethylsilane, Pd(PPh3)4, CuI, Et3N, DMF, rt, 83%. (c) TBDMSCl, AgNO3, base, THF, rt, 36% (4), 40% (5). (d) 2-cyanoethyl-(N,N-diisopropylamino)chlorophosphite, DIPEA, THF, rt, 70% (6), 84% (7).
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