Synthesis of Abasic Locked Nucleic Acid and Twoseco-LNA Derivatives and Evaluation of Their Hybridization Properties Compared with Their More Flexible DNA Counterparts

Abstract

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.

Gapmer Antisense Oligonucleotides Containing 2′,3′‐Dideoxy‐2′‐fluoro‐3′‐C‐hydroxymethyl‐β‐d‐lyxofuranosyl Nucleotides Display Site‐Specific RNase H Cleavage and Induce Gene Silencing

“…For synthesis of ONs containing monomers X and Y, polystyrene Glenn Unysupport (Nr.2 6-5040-10) and inverted DNA phosphoramidites were used. Phosphoramidites 1 and 2 were incorporated by am anual hand-coupling procedure [23] by using Activator 42 (0.25 m,5 -[3,5-bis(trifluoromethyl)phenyl]-1 H-tetrazole) as activator and extended coupling time (16 min), resulting in stepwise coupling yields of > 97 %f or phosphoramidites 1 and 2. Cleavage from the solid support and removal of nucleobase protecting groups were performed by using 28 %a queous ammonia (12 h, RT).A fter evaporation of all solvents, detritylation was performed by using an 80 %a queous solution of acetic acid (20 min, RT),w hich was followed first by desalting by using an aqueous solution of sodium acetate (3 m,1 5mL) and sodium perchlorate (5 m, 15 mL) and then by addition of ice-cold (store at À20 8C) acetone.…”

“…26‐5040‐10) and inverted DNA phosphoramidites were used. Phosphoramidites 1 and 2 were incorporated by a manual hand‐coupling procedure by using Activator 42 ® (0.25 m , 5‐[3,5‐bis(trifluoromethyl)phenyl]‐1 H ‐tetrazole) as activator and extended coupling time (16 min), resulting in stepwise coupling yields of >97 % for phosphoramidites 1 and 2 . Cleavage from the solid support and removal of nucleobase protecting groups were performed by using 28 % aqueous ammonia (12 h, RT).…”

Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2′-Amino-LNA Functionalized with Galactose Units

“…ONs were synthesized on a DNA synthesizer (PerSpective Biosystems Expedite 8909, (Framingham, MA, USA)) in 1.0 μmol scale using manufacturer’s standard protocols. For incorporation of monomer M 1 [ 56 ] a hand-coupling procedure [ 68 ] was used (20 min coupling time and 5-[3,5-bis(trifluoromethyl)phenyl]-1 H -tetrazole (0.25 M, in anhydrous CH 3 CN) as activator). The coupling efficiencies of standard DNA phosphoramidites and phosphoramidite 10 based on the absorbance of the dimethoxytrityl cation released after each coupling varied between 95% and 98%.…”

Novel conformationally constrained 2′-C-methylribonucleosides: synthesis and incorporation into oligonucleotides

“…The 2'-C-Me-LNA and 2'-C-Me-ONA phosphoramidites 9a and 9b, respectively, were successfully used in automated ON synthesis on a commercial nucleic acid synthesizer. The stepwise coupling yields were ∼80% for the 2'-C-Me-LNA phosphoramidite 9a employing "hand coupling conditions" 17 and ∼99% for standard DNA and LNA phosphoramidites.…”

“…These adopt a 2'-endo (South-type) pseudorotational conformation, and when incorporated into oligonucleotides (phosphodiester 2'-5'-linkage) they show a selectivity for RNA over DNA and a significant decrease in thermal stability against complementary DNA and RNA. 17 It is also 3 relevant to mention that several 2'-C-methylribonucleosides are selective inhibitors of HCV replication including the FDA approved drug Sofosbuvir as the most prominent compound. 18 Herein we describe the synthesis of two novel constrained ribonucleotide phosphoramidites bearing a 2'-C-methyl substituent and either a O2'-C4' (LNA-analogue) or a O3'-C4' (ONAanalogue) methylene bridge.…”