An Exocyclic Methylene Group Acts As a Bioisostere of the 2′-Oxygen Atom in LNA

Abstract

We show for the first time that it is possible to obtain LNA (Locked Nucleic Acid 1) like binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2’-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2’-position and a propynyl group at C-4’ position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2’-deoxyribose residue of the 5’-terminal G of a neighboring duplex, resulting in the formation of a CH…O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2’-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA ASOs showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moeities in nucleic acid structure and emphasizes greater use of this bio-isostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

Synthesis, Duplex Stabilization and Structural Properties of a Fluorinated Carbocyclic LNA Analogue

“…10 Interestingly, the cLNA analogue 8 , in which the 2′-oxygen atom was replaced with a exocyclic methylene group, showed LNA-like duplex stabilization properties while the corresponding saturated analogues ( 9 and 10 ) were less stabilizing. 11 Using crystal structure data, we showed that the exocyclic methylene group retains net negative electrostatic potential at the brim of the minor groove and participates in CH···O type interactions with acceptor molecules in the minor groove of the modified duplex. In contrast, the saturated cLNA analogues 9 and 10 were unable to extend the water of hydration network around the backbone phosphates and the nucleobases resulting in diminished duplex stabilizing properties.…”

“…11 Attempts to cleave the exocyclic double bond in 11 using osmium tetroxide and sodium periodate gave poor conversions to the intermediate ketone. Presumably, the bulky tert -butyldiphenylsilyl (TBDPS) and 2-naphthylmethyl (Nap) protecting groups on the 5′- and the 3′-hydroxyl groups create steric crowding and limit access of the catalyst to the exocyclic methylene group in 11 .…”

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

“…The success of this reaction was recently exploited for the synthesis of methylene-carba-LNA by Seth et al (see Section 2.5). 40 Another type of bicyclic nucleoside synthesized through freeradical cyclization is 3′,4′-β-fused six-membered bicyclic nucleosides 31 (Scheme 4). 48 In the radical precursors 29, an allyl is attached to the 5′-O through an ether bond.…”

“…40 Such an intramolecular addition of the C2′ radical to a C3′-Otethered alkyne, generating a 2′,3′-fused five-membered heteroring with exocyclic olefin function (28, Scheme 3), has been established in 1991 in the Uppsala lab. 44 The synthesis of methylene-carba-LNA started from 5′-TBDPS-3′-(2-methylnapthalene)-allo-furanose derivative 108 (Scheme 12).…”

Synthesis and biological evaluation of 2′,4′- and 3′,4′-bridged nucleoside analogues

“…Following the synthesis and evaluation of the 2′- O ,4′-C- 4 and 3′- O ,4′-C- 5 bridged nucleic acids, there have been reports on the synthesis of 2′- NO ,4′- C systems, 6 2′- N ,4′- C -bridged systems, 7 2′,4′-propylene-BNAs, 8 carbocyclic-BNAs, 9 and more. 10 However, only a few groups have explored the biological activities of bridged nucleoside analogues themselves (see Figure 2 for selected examples).…”