Synthesis of Conformationally Locked Carba-LNAs through Intramolecular Free-Radical Addition to C═N. Electrostatic and Steric Implication of the Carba-LNA Substituents in the Modified Oligos for Nuclease and Thermodynamic Stabilities

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Abstract
The syntheses of the hitherto unavailable parent fully unsubstituted carba-LNA and its C7′-amino and/or C6′-hydroxyl substituted derivatives, have been accomplished by the intramolecular 5-exo free-radical addition to a C4′-tethered C═N to give carba-LNAs with variable hydrophilic substituents at C6′/C7′ (amino and/or hydroxyl). They have been introduced into isosequential antisense oligonucleotides (AONs) to examine how their relative electrostatic and steric effects in the minor groove of a putative AON-RNA duplex affect the target affinity, nuclease resistance, and RNase H elicitation. We show that 2′-oxygen in LNA is important in stabilizing the DNA/DNA and DNA/RNA duplexes vis-à-vis the unsubstituted carba-LNA and its other derivatives and that hydrophobic groups at C6′/C7′ in both carba-LNA and carba-ENA relatively destabilize the AON/DNA duplex more profoundly than those in the AON/RNA duplexes. Two main factors affect the relative stabilities of AON/DNA versus AON/RNA duplexes: (i) hydration in the minor groove depending upon hydrophilicity vis-à-vis hydrophobicity of the substituents, and (ii) the relative size of the minor groove in the AON/DNA versus AON/RNA duplexes dictates the steric clash with the substituents depending upon their relative chiralities. We also show how the chirality and chemical nature of the C6′/C7′ substituents affect the nuclease stability as well as the thermal stability and the RNase recruitment by AON/RNA duplexes.

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