Nicolai K. Andersen

Synthesis of 5‐(1,2,3‐Triazol‐4‐yl)‐2′‐deoxyuridines by a Click Chemistry Approach: Stacking of Triazoles in the Major Groove Gives Increased Nucleic Acid Duplex Stability

A general protocol for converting alkyl and aryl halides into azides and for converting these in situ into 1,4‐disubstituted triazoles was applied with 5‐ethynyl‐2′‐deoxyuridine. This afforded three modified 2′‐deoxyuridine analogues with either unsubstituted or 1‐phenyl‐/1‐benzyl‐substituted triazoles in their 5‐positions. Modelling demonstrates coplanarity of the two heteroaromatic rings, and UV spectroscopy showed the uracil pKa values to be almost unchanged. The three nucleosides were introduced into nonamer oligonucleotides by phosphoramidite chemistry. The heteroaromatic triazoles became positioned in the major grooves of the short dsDNA and DNA–RNA duplexes. While single modifications led to decreased duplex stability, the stacking of four consecutive modifications led to enhanced duplex stability, especially for DNA–RNA duplexes. The duplex structures were studied by CD spectroscopy and molecular dynamics simulations, which supported the conjecture that the duplex stabilizing effect is due to efficient stacking of the heteroaromatic triazoles.

Efficient RNA-targeting by the introduction of aromatic stacking in the duplex major groove via 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridines

Three pyrimidine nucleosides with differently substituted phenyltriazoles attached to the 5-position were prepared by Cu(I)-assisted azide-alkyne cycloadditions (CuAAC) and incorporated into oligonucleotides. Efficient π-π-stacking between two or more phenyltriazoles in the major groove was found to increase the thermal stability of a DNA:RNA duplex significantly. The best stacking, and most stable duplex, was obtained by a sulfonamide substituted derivative.

Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson–Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson–Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2′-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

N2′-Functionalized 2′-Amino-α-L-LNA Adenine Derivatives—Efficient Targeting of Single Stranded DNA

The synthesis of two pyrene-functionalized 2′-amino-α-L-LNA adenine building blocks is outlined and initial results from thermal denaturation studies are presented.

A click chemistry approach towards nucleic acid major groove functionalization

A synthetic strategy towards new aromatic nucleoside derivatives introducing additional aromatic functionality placed in the major groove of a modified DNA duplex is presented. The functionalities are introduced using Click Chemistry conditions and found to increase the overall duplex stability.