Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

The azides 1 and 2 bearing a phenoxazinium and a coumarin fluorophore, respectively, were applied in postsynthetic “click”-type bioconjugation and coupled to oligonucleotides modified with alkyne groups using two alternative approaches: (i) as a nucleotide modification at the 2′-position of uridine and (ii) as a nucleotide substitution using (S)-(−)-3-amino-1,2-propanediol as an acyclic linker between the phosphodiester bridges. The corresponding alkynylated phosporamidites 3 and 6 were used as DNA building blocks for the preparation of alkyne-bearing DNA duplexes. The base pairs adjacent to the site of modification and the base opposite to it were varied in the DNA sequences. The modified duplexes were investigated by UV/vis absorption spectroscopy (including melting temperatures) and fluorescence spectroscopy in order to study the different optical properties of the two chromophores and to evaluate their potential for bioanalytical applications. The sequence-selective fluorescence quenching of phenoxazinium 1 differs only slightly and does not depend on the type of modification, meaning whether it has been attached to the 2′-position of uridine or as DNA base surrogate using the acyclic glycol linker. The 2′-chromophore-modified uridine still recognizes adenine as the counterbase, and the duplexes exhibit a sufficient thermal stability that is comparable to that of unmodified duplexes. Thus, the application of the 2′-modification site of uridine is preferred in comparison to glycol-assisted DNA base surrogates. Accordingly, the coumarin dye azide 2 was attached only to the 2′-position of uridine. The significant Stokes shift of ∼100 nm and the good quantum yields make the coumarin chromophore a powerful fluorescent label for nucleic acids.