Conjugation of N-(3-(9-Ethynyl-6H-indolo[2,3-b]quinoxalin-6-yl)propyl)-2,2,2-trifluoroacetamide Intercalator to a Triplex Forming Oligonucleotide, a Three-Way Junction, and a G-Quadruplex

Abstract

A new intercalating nucleic acid monomer Z comprising an 3-(9-((4-oxyphenyl)ethynyl)-6H-indolo[2,3-b]quinoxalin-6-yl)propan-1-amine moiety was synthesized. When Z was inserted into triplex forming oligonucleotides, high thermal stability was observed for its corresponding Hoogsteen-type triplexes. Three-way junction (TWJ) was studied by targeting a DNA strand to the foot of a DNA or an RNA hairpin. When Z was inserted into the DNA strand, this resulted in the highest increase of thermal melting ever reported for a TWJ modified by insertion of an intercalator into the junction site. Experiments with mismatches confirmed formation of the TWJ. Improvements in stability of a G-quadruplex were achieved by insertion of the monomer Z by replacement of one of the nucleotides in the TGT loop. The first steps in the synthesis of the monomer Z were condensation of 5-iodoisatin with o-phenylene diamine under reflux in acetic acid and subsequent alkylation reaction with 2-(3-bromopropyl)isoindoline-1,3-dione followed by suitable reduction led to 3-(9-Iodo-6H-indolo[2,3-b]quinoxalin-6-yl)propan-1-amine which in turn was treated with ethyl trifluoroacetate with formation of 2,2,2-trifluoro-N-(3-(9-iodo-6H-indolo[2,3-b]quinoxalin-6-yl)propyl)acetamide. This compound was reacted by Sonogashira coupling conditions to give the diol which was converted into the DMT-protected phosphoramidite, which in turn was used to incorporate the monomer Z into oligonucleotides.