Synthesis and Biophysical Evaluation of 2′,4′-Constrained 2′O-Methoxyethyl and 2′,4′-Constrained 2′O-Ethyl Nucleic Acid Analogues

Abstract

We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.

Pharmacology of a Central Nervous System Delivered 2′-O-Methoxyethyl–Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates

“…The binding affinity of an ASO to its target can be increased dramatically by the incorporation of 29,49-bridged nucleic acid modifications, such as locked nucleic acid (Vester and Wengel, 2004) and 29,49-constrained 29-O-ethyl (cEt) (Seth et al, 2010) (Fig. 3A).…”

Synthetic CRISPR RNA-Cas9–guided genome editing in human cells

“…The 2′-F binding is largely energetically driven by the electronegative substituent (33). Alternatively, affinity, as well as stability, can be increased with the use of constrained bicyclic analogs like the cEt substitution that links the 2′ and 4′ positions of the ribose sugar (34). Using the same experimental design as in Fig.…”

Enhanced splice correction by 3′, 5′-serinol and 2′-(ω-O-methylserinol) guarded OMe-RNA/DNA mixmers in cells

“…21 Such AONs also show reduced binding efficiency to RNA. The enzyme resistant LNA analogs 22,23 such as c-OMe or c-Et also require several synthetic steps and separation of diastereomers during their synthesis. These shortcomings are indicative of the pressing need for efficient AON analogs that employ relatively simple chemistry, are chirally homogeneous but are still endowed with less toxic off-target effects and have higher efficiencies.…”