Tuning of conformational preorganization in model 2',5'- and 3',5'-linked oligonucleotides by 3'- and 2'-O-methoxyethyl modification

Abstract

Conformational properties of trimeric and tetrameric 2',5'-linked oligonucleotides, 3'-MOE-A3(2',5') (1) and 3'-MOE-A4(2',5') (2), and their 3',5'-linked analogs, 2'-MOE-A3(3',5') (3) and 2'-MOE-A4(3',5') (4), were examined with the use of heteronuclear NMR spectroscopy. The temperature-dependent 3JHH, 3JHP and 3JCP coupling constants, acquired in the range of 273-343 K, gave insight into the conformation of sugar rings in terms of a two-state North <---> South (N <---> S) pseudorotational equilibrium and into the conformation of the sugar-phosphate backbone in the model antisense oligonucleotides 1-4. 2',5'-linked oligomers 3'-MOE-A3(2',5') (1) and 3'-MOE-A4(2',5') (2) show preference for N-type conformers and indication of A-type conformational features, which is prerequisite for antisense hybridization. The drive of N <---> S equilibrium in 1-4 has been rationalized with the competing gauche effects of 2'/3'-phosphodiester and 3'/2'-MOE groups, anomeric and steric effects. Furthermore, the pairwise comparisons of 3'-MOE with 3'-OH and 3'-deoxy 2',5'-linked adenine trimers emphasized the fine tuning of N <---> S equilibrium in 3'-MOE-A3(2',5') (1) and 3'-MOE-A4(2',5') (2) by the steric effects of 3'-MOE group and the possibility of water-mediated H-bonds with vicinal phosphodiester functionality. In full correspondence, the drive of N <---> S equilibrium towards N by 2'-MOE in 3',5'-linked analogs 2'-MOE-A3(3',5') (3) and 2'-MOE-A4(3',5') (4) is weaker in comparison with 3'-OH group in the corresponding ribo analogs. Beta(t), gamma+ and epsilon- rotamers are preferred in both 2',5'- and in 3',5'-linked oligonucleotides 1-4.

Scheme 1. (A) Non-terminal monomer building block of 2′,5′-linked trinucleoside diphosphate 3′-MOE-A₃^2′,5′ (1) and tetranucleoside triphosphate 3′-MOE-A₄^2′,5′ (2). (B) Non-terminal monomer building block of 3′,5′-linked trinucleoside diphosphate 2′-MOE-A₃^3′,5′ (3) and tetranucleoside triphosphate 2′-MOE- A₄^3′,5′ (4).

Scheme 1. (A) Non-terminal monomer building block of 2′,5′-linked trinucleoside diphosphate 3′-MOE-A₃^2′,5′ (1) and tetranucleoside triphosphate 3′-MOE-A₄^2′,5′ (2). (B) Non-terminal monomer building block of 3′,5′-linked trinucleoside diphosphate 2′-MOE-A₃^3′,5′ (3) and tetranucleoside triphosphate 2′-MOE- A₄^3′,5′ (4).

Figure 1

The populations of North conformers as a function of temperature in individual residues in (A) 3′-MOE-A₃^2′,5′ (1), (B) 3′-MOE-A₄^2′,5′ (2), (C) 2′-MOE-A₃^3′,5′ (3) and (D) 2′-MOE-A₄^3′,5′ (4). Straight lines are the best fits through experimental data points. Labels on the straight lines indicate residue numbers.

Figure 2

The experimental thermodynamic parameters for N ↔ S equilibrium [ΔH⁰ (dashed bars), –TΔS⁰ at 298 K (gray bars), ΔG⁰ (white bars)] in various adenosine trimers. Individual residues in oligonucleotides are denoted with A1–A4 beginning from the 5′-terminal end. A positive sign of ΔH⁰, –TΔS⁰ and ΔG⁰ denotes a drive of the N ↔ S pseudorotational equilibrium towards the North-type conformers, whereas the negative sign denotes the drive towards South-type conformers. The values of ΔH⁰ and ΔS⁰ were calculated from slopes and intercepts of van’t Hoff plots according to the relation: ln (X_S/X_N) = –(ΔH⁰/R)(1000/T) + ΔS⁰/R. The Pearson correlation coefficients of van’t Hoff plots were –0.995 for A1 in 1, –0.990 for A2 in 1, –0.811 for A3 in 1, –0.991 for A1 in 2, –0.993 for A2 in 2, –0.974 for A3 in 2 and –0.992 for A4 in 2. Thermodynamic quantities for ApApA and 3′d(A2′p5′A2′p5′A) were calculated from reported populations (27,40).

Figure 3

Relative orientations of adenine, phosphodiester functionality, 3′-MOE group and other substituents in (A) N- and (B) S-type conformers of non-terminal residues in 2′,5′-linked oligonucleotides 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2). The 3′-MOE group adopts pseudoequatorial orientation in the N-type conformation (A). In addition, oxygen atoms of 3′-MOE group which adopt gauche orientation are predisposed for water-mediated H-bonding interactions with 2′-phosphodiester functionality. In S-type conformation the 3′-MOE group adopts less favorable pseudoaxial orientation (B).