Synthesis and Antisense Properties of 2'β-F-Arabinouridine Modified Oligonucleotides with 4'- C-OMe Substituent

Abstract

A novel 2'-F,4'-C-OMe⁻arabinouridine (araU) was successfully synthesized and introduced into oligonucleotides. The oligonucleotide containing 2'-F,4'-C-OMe⁻araU exhibited improved nuclease resistance and RNA hybridizing selective ability relative to 2'-F⁻araU. In particular, when 2'-F,4'-C-OMe⁻araU inserted into C⁻H⋯F⁻C bonding-favorable 5'⁻uridine⁻purine⁻3' steps, the modified oligonucleotide showed remarkable binding affinity and selectivity to RNA complements. Thus, 2'-F,4'-C-OMe⁻araU has valuable antisense properties and can be used as novel chemical modification for antisense therapeutic strategy.

Keywords: arabinonucleotide; chemical modification; fluorine; pseudohydrogen bond.

Figure 1

Structure of fluorinated nucleotide 2′-F–arabinonucleic acid (ANA), 2′-F–RNA, selected 4′-substituted 2′-F–ANA derivatives, and 2′-F,4′-C-OMe–arabinouridine (araU) designed in the present study. B = nucleobase, U = uracil-1-yl.

Scheme 1

The general synthesis procedure of 2′-F,4′-C-OMe–araU phosphoramidite 8. Reagents and conditions: (i) imidazole, Ph₃P, I₂, tetrahydrofuran (THF), 0 °C–room temperature (rt), 4 h, 78%; (ii) NaOMe–MeOH, reflux, 3 h, 85%; (iii) methanol, PbCO₃, I₂, THF (dry), 0 °C–rt, 3 h, 90%; (iv) benzoyl chloride (BzCl), pyridine (dry), dichloromethane (DCM), 0 °C–rt, 3 h, 95%; (v) (a) m-chloroperbenzoic acid (m-CPBA), DCM/H₂O, 0 °C–rt, 3 h, (b) NH₃–MeOH, rt, 12 h, 64% two steps; (vi) dimethoxytrityl chloride (DMTrCl), pyridine (dry), rt, 12 h, 81%; (vii) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite, 1H-tetrazole, DCM (dry), room temperature, 3 h, 74%.

Figure 2

The circular dichroism (CD) spectra of duplexes formed by the modified oligonucleotides ON2–ON5 with RNA complements. Spectra were recorded three times independently at 20 °C after annealing the duplexes under the same condition described in ultraviolet (UV) melting experiments.

Figure 3

Enzymatic stability of 5′–d(TTTTTTTTUT)–3′ against snake venom phosphodiesterase (SVPDE). U = 2′-F,4′-C-OMe–araU (X, ON12, cross), 2′-F–araU (Z, ON13, triangle), 3′-phosphorothioate-T (Ts, ON14, rectangle), and natural DNA–dT (ON15, diamond). Degradation of oligonucleotides (17.5 pM) by SVPDE (0.02 μg/mL) was performed in a buffer (pH 8.0) containing 10 mM MgCl₂, 50 mM Tris-HCl at 37 °C.