Synthesis of DNA Building Blocks With Aminonaphthalimides and Cyanovinylenes as Uncharged Fluorophores: Fluorescent Readout From Turn‐On to Red‐Shift

Three phosphoramidites of aminonaphthalimides and two of cyanovinylenes were synthesized as DNA building blocks and incorporated as novel fluorescent nucleotide analogs into DNA. The chromophores were attached to oligonucleotides using propanediol as acyclic linker between the phosphodiester bridges. This linker provides high stability and flexibility in comparison to the conventional 2-deoxyfuranoside; it places the chromophore as nucleotide analog into DNA and is compatible with automated oligonucleotide chemistry. For aminonaphthalimides, two different linker lengths were compared, and the role of demethylation at the exocyclic amino group was evaluated. For cyanovinylenes, the role of the alkylamino group for fluorescence response was evaluated. The exploration of the optic-spectroscopic properties of the dyes in different DNA architectures with the possibility to form interstrand and intrastrand dimers showed that aminonaphthalimides and cyanovinylenes can be used as nucleic acid-sensitive dyes by their fluorescence turn-on and red-shift as readouts. The fluorescence turn-on was tested by probing N$^6$-methyl-2′-deoxyadenosine (m$^6$A) in DNA.