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

• Published in: Bioconjugate chemistry Vol. 21; no. 9; pp. 1629 - 1641
• Main Authors: Seela, Frank, Pujari, Suresh S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.09.2010
• Subjects: Adenine - analogs & derivatives; Adenine - chemistry; Alkalies; Alkynes - chemistry; Azides - chemistry; Base Pairing; Chemical synthesis; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA, Single-Stranded - chemistry; Dyes; Fluorescence; Fluorescent Dyes - chemistry; Molecular chemistry; Molecular Dynamics Simulation; Nucleic Acid Denaturation; Oligonucleotides - chemical synthesis; Oligonucleotides - chemistry; Organophosphorus Compounds - chemical synthesis; Organophosphorus Compounds - chemistry; Spectrometry, Fluorescence; Staining and Labeling; Temperature
• DOI: 10.1021/bc100090y
• ISSN: 1043-1802

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.