Azide−Alkyne “Click” Conjugation of 8-Aza-7-deazaadenine-DNA: Synthesis, Duplex Stability, and Fluorogenic Dye Labeling

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Abstract

The internal dye labeling of DNA by the Huisgen-Meldal-Sharpless "click" reaction is described. Fluorogenic 9-azidomethyl anthracene 2 and 3-azido-7-hydroxycoumarin 3 were employed in the postsynthetic functionalization of oligonucleotides incorporating octa-(1,7)-diynyl-8-aza-7-deaza-2'-deoxyadenosine 1. Nucleoside 1 was prepared by Sonogashira cross coupling from the corresponding 7-iodo compound, converted into the corresponding phosphoramidite, and oligonucleotides were synthesized. To evaluate the influence of ligands on the oligonucleotide duplex stability, benzyl azide 4 (nonpolar), and 2',3'-dideoxy azidothymidine 5 (AZT) (polar) were introduced along with the fluorogenic dyes 2 and 3. DNA duplexes with octa-1,7-diynyl side chains (i.e., containing 1) are more stable than oligonucleotides containing 8-aza-7-deaza-2'-deoxyadenosine, unveiling that this side chain has steric freedom. A single conjugation by an anthracene residue led to a 9 °C T(m) increase of duplex melting. Contrary to 7-deazaadenine dye conjugates, the 8-aza-7-deazaadenine conjugates show virtually no fluorescence quenching, thereby developing almost as strong fluorescence as side chain click derivatives (32 and 33) in the absence of 8-aza-7-deazaadenine moiety. Duplexes containing the 8-aza-7-deazaadenine dye conjugate show increased fluorescence over single-stranded DNA. Mismatches with dA, dG, and dC develop reduced fluorescence compared to the fully matched base pair. Molecular dynamics simulations revealed that the bulky dye molecules are accommodated well in duplex DNA.

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