Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications

Abstract

Antisense oligonucleotides (ASOs) are becoming important drugs for hard to treat diseases. Modifications to their DNA backbones are essential to inhibit degradation in vivo, but they can reduce binding affinity to RNA targets. To address this problem we have combined the enzymatic resistance of carbamate (CBM) DNA backbone analogues with the thermodynamic stability conferred by locked nucleic acid sugars (LNA). Using a dinucleotide phosphoramidite strategy and automated solid phase synthesis, we have synthesised a set of oligonucleotides modified with multiple LNA-CBM units. The LNA sugars restore binding affinity to RNA targets, and in this respect LNA position with respect to the CBM linkage is important. Oligonucleotides containing carbamate flanked on its 5'and 3'-sides by LNA form stable duplexes with RNA and unstable duplexes with DNA, which is desirable for antisense applications. Carbamate-LNA modified oligonucleotides also show increased stability in the presence of snake venom and foetal bovine serum compared to LNA or CBM backbones alone.

Fig. 1. Carbamate-modified DNA backbones containing LNA sugars. Set 1 (A–D) and set 2 (E, F) comprise of dimers with the constitutional carbamate isomers CBM₁ and CBM₂ respectively. A = DNA-CBM₁-DNA, B = DNA-CBM₁-LNA, C = LNA-CBM₁-DNA, D = LNA-CBM₁-LNA, E = DNA-CBM₂-DNA, F = DNA-CBM₂-LNA.

Scheme 1. Synthesis of CBM₁-linked dinucleotides 13–16 using different combinations of 3′-activated carbonates 2, 4 and 5′-amines 5, 8. Reagents and conditions (i) p-nitrophenyl chloroformate, pyridine, 80 °C, 20 h, crude 2 60%; (ii) CDI, THF, rt, 20 h, crude 4 84%; (iii) NaN₃, DMF, 65 °C, 20 h, 7 90%; (iv) H₂, Pd(OH)₂ (20%)/C MeOH, rt, 3 h; NH₄HCO₂, reflux, 4 h, 8 87%; (v) DMAP, pyridine, 80 °C, 20 h, 9 62%, 10 51%, 11 63%, 12 70%; (vi) Chloro(diisopropylamino)-β-cyanoethoxyphosphine, DIPEA, CH₂Cl₂, rt, 1 h, 13 54%, 14 57%, 15 36%, 16 88%.

Scheme 2. Synthesis of CBM₂ dinucleotides 24, 27 using activated 3′-amine 17 and 5′-activated carbonates 19, 21. Reagents and conditions (i) p-nitrophenyl chloroformate, pyridine, 80 °C, 20 h, 19 76%, 21 crude 73%, (ii) HOBt, pyridine, 80 °C, 20 h, 22 67%, 25 63%; (iii) TBAF, THF, 0 °C, 2 h, 23 77%; (iv) Pd(OH)₂ (20%)/C, NH₄HCO₂, 65 °C, 2 h, 26 97%; (v) Chloro(diisopropylamino)-β-cyanoethoxyphosphine, DIPEA, CH₂Cl₂, rt, 1 h, 24 80%, 27 43%.

Fig. 2. (Top) UV-melting data, 3 μM of oligonucleotides were added to 10 mM phosphate, 200 mM NaCl buffer, pH 7.0 and heated from 20–85 °C. T_m values were determined from an average of six ramps from a smoothed plot of dA/t. (A) First derivative T_m curves against DNA targets containing triple modifications of CBM-LNA, (B) First derivative T_m curves against RNA targets containing triple modifications of CBM-LNA. (Bottom) Circular dichroism spectra of 3 μM oligonucleotides in 10 mM phosphate, 200 nM NaCl buffer, pH 7.0. Spectra were averaged from four scans and smoothed to 20 points using a third order polynomial. (C) Triple incorporation of CBM-LNA modifications against DNA targets, (D) Triple incorporation of CBM-LNA modifications against RNA targets. Overall duplex conformation against both targets remained largely unchanged. Against DNA targets hypsochromic shifts were observed with addition of LNA.

Fig. 3. (Top) 20% denatured polyacrylamide gel electrophoresis (PAGE) results from snake venom 3′-exonuclease assay. Samples were incubated in 50 mM Tris-HCl, 10 mM MgCl₂ buffer pH 9.0 at 37 °C. Aliquots were removed at t = 0, 5, 15, 30 and 60 min and frozen before analysis. (A) Gel showing unmodified and LNA controls, (B) gel showing oligonucleotides with carbamate and combined LNA-carbamate modification. ON14 GCTT^LGCTT^LCGTT^LCC, ON15 GCT^LT^LGCT^LT^LCGT^LT^LCC, ^L = LNA. Results show increased nuclease resistance when introducing three separate 5′/3′-LNA-CBM₁ modifications (ON11). (Bottom) 20% denatured polyacrylamide gel electrophoresis (PAGE) results from FBS assay of modified templates containing triple incorporation of CBM₁-LNA dinucleotides. Samples were incubated in Dulbecco's PBS buffer at 37 °C. Aliquots were removed at t = 0, 4, 8, 12 and 24 h and frozen before analysis. (C) Gel showing unmodified and LNA controls, (D) gel showing oligonucleotides with carbamate and combined LNA-carbamate modification. Combined 5′/3′-LNA and CBM₁ modifications (ON11) showed increased serum resistance compared to unmodified control.