Synthesis of DNA Modified with Boronic Acid: Compatibility to Copper(I)-Catalyzed Azide–Alkyne Cycloaddition

The postsynthetic and sequence-specific ligation chemistry of a phenylboronic acid to oligonucleotides using the amide bond formation was worked out. In the first coupling experiments with 4-carboxyphenylboronic acid, a 5′-hexylamino-modified oligonucleotide was used to evaluate and optimize the reaction conditions. This postsynthetic modification works best in the presence of TBTU and triethanolamine and in a degassed DMF/carbonate buffer solvent mixture. The successful attachment of the boronic acid was evidenced by HPLC separation from phenol side products and clear identification via MALDI-TOF mass spectrometry as a citric acid derivative. This postsynthetic chemistry was further combined with the established Cu­(I)-catalyzed azide–alkyne cycloaddition chemistry to allow the first orthogonal and postsynthetic incorporation of both the phenylboronic acid moiety and two different cyanine-styryl dyes. Because of the undesired reactivity of boronic acids by the presence of copper salts, the dye azides were first attached to the presynthesized oligonucleotides using the Cu­(I)-catalyzed cycloaddition at the 2′-position of a propargylated uridine. After careful removal of all copper contaminants, the amide bond with the 4-carboxyphenylboronic acid at the propylamine linker of a 7-deaza-2′-deoxyadenosine as anchor point was formed. These doubly modified oligonucleotides were characterized by their optical properties to elucidate the influence of the phenylboronic acid. The latter modification has only little influence on the fluorescence of the applied dyes. In conclusion, this postsynthetic and orthogonal chemistry opens the way to a broad variety of applications, in particular, saccharide detection based on fluorescent DNA aptamers.