Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bc(en)-T and iso-tricyclo-T nucleosides

Abstract

We present the synthesis of the two novel nucleosides iso-tc-T and bc(en)-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6'-C7' within the carbocyclic ring is planarized by either the presence of a C6'-C7' double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2'-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bc(en)-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3'-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that T m data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Keywords: DNA/RNA affinity; X-ray structures; nucleic acids; nucleosides; oligonucleotide therapy; oligonucleotides.

Figure 1

Chemical structures and carbon numbering scheme of tricyclo(tc)-DNA (top, left), bicyclo(bc)-DNA (top, right) and the newly synthesized iso-tricyclo(iso-tc)-DNA (bottom, left) and bicyclo-en(bc^en)-DNA (bottom, right).

Scheme 1

Conditions: (a) NaBH₄, CeCl₃·7H₂O, MeOH, −78 °C → rt, 1.5 h, 73% (+9% of C6-epimer); (b) TBS-Cl, imidazole, CH₂Cl₂, rt, 16 h, 79%; (c) Et₂Zn in hexane (1 M), CH₂I₂, CH₂Cl₂, 0 °C → rt, 16 h, 86%; (d) PivCl, DMAP, pyridine, ClH₂C–CH₂Cl, 70 °C, quant.; (e) TBAF, THF, rt, 19 h, 95%; (f) thymine, BSA, SnCl₄, CH₃CN, 0 °C → rt, 17 h, 56% 7β + 22% (7α/β 2.5:1); (g) Bu₄NOH, H₂O/dioxane, rt, 16 h, 94%; (h) DMTrCl, pyridine, CH₂Cl₂, rt, 24 h, 98%; (i) CEP-Cl, DIPEA, THF, rt, 4 h, 89%.

Scheme 2

Conditions: (a) thymine, BSA, TMSOTf, TMSCl, CH₃CN, rt, 2.5 h; (b) DMTrCl, pyridine, rt, 16 h, 29% of 12α and 34% of 12β (over two steps); (c) CEP-Cl, DIPEA, THF, rt, 1 h, 94%.

Figure 2

X-ray structure of top row: nucleosides 8β (left), 11β (center) and overlay of both structures (right); bottom row: tc-T (Mol A, left, Mol B, right).

Scheme 3

Pathways for elimination of the modified nucleotides during the oxidation step in oligonucleotide assembly.