Chiral-4′-Methyl-α‑(l)‑Threofuranosyl Nucleic Acids: Synthesis, Structure, Binding Affinity, Nuclease Resistance, and Polymerase Recognition

Abstract

α-(l)-(3′–2′)-Threofuranosyl nucleic acid (TNA) cross-pairs with DNA and RNA and is not degraded by nucleases and thus has potential for use in diagnostics and therapeutics based on nucleic acids. Here, we describe the synthesis of 15 building blocks of chiral 4′-(R)- and 15 building blocks of chiral (S)-methyl-α-(l)-threofuranosyl nucleic acid (4′-Me-TNA) monomers and their incorporation into oligonucleotides. Crystallographic studies of RNAs containing 4′-Me-TNA modifications revealed the impact of the methyl substituent on the sugar pucker and duplex conformation. Biostability studies showed that a terminal 4′-Me-TNA modification on a deoxyoligonucleotide imparted resistance to nuclease digestion equivalent to unmethylated TNA, which is markedly superior to that of more conventional 2′-ribo and 2′-deoxy modifications. A single nucleotide incorporation of the 4′-(R)-Me-TNA destabilized an otherwise all-RNA 12-mer duplex by 6.5 to 12 °C, depending on the base, relative to the unmodified RNA control duplex; the (S) isomer was less destabilizing than the (R) isomer. Computational models were used to rationalize the high levels of nuclease resistance and the thermal destabilization. The 4′-Me-TNA triphosphates are poorly recognized by an engineered TNA polymerase making it unlikely that the 4′-Me-TNA triphosphates will be recognized by natural polymerases. Together, these properties warrant the evaluation of 4′-Me-TNA in nucleic acid-based therapeutics.