Solvatochromic fluorescent 4-aminophthalimide (API) and 4-(N,N-dimethylamino)phthalimide (DAPI) were attached covalently to 2′-deoxycytidine or -adenosine via a non-conjugated propargyl linker by Sonogashira cross-coupling reactions of N-propargylphthalimides with halogenated nucleosides. The nucleosides were phosphorylated to triphosphates and enzymatically incorporated into oligonucleotides by DNA polymerases. API-labelled DNA was used for the detection of DNA protein interactions with either the sequence-specific p53 protein or a non-specific single strand binding (SSB) protein. Both proteins changed the polarity around the fluorophore and increased (2–3 fold) the intensity of API fluorescence.
