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 bioconjugationand coupled to oligonucleotides modified with alkynegroups using two alternative approaches: (i) as a nucleotide modification at the 2′-position of uridineand (ii) as a nucleotide substitution using (S)-(−)-3-amino-1,2- propanediolas an acyclic linker between the phosphodiester bridges. The corresponding alkynylatedphosporamidites 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 spectroscopyin 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 phenoxazi...