Diene-modified nucleotides for the Diels-Alder-mediated functional tagging of DNA.
We explore the potential of the Diels-Alder cycloaddition for the functional tagging of DNA strands. A deoxyuridine triphosphate derivative carrying a diene at position 5 of the pyrimidine base was synthesized using a two-step procedure. The derivative was efficiently accepted as substrate in enzymatic polymerization assays. Diene carrying strands underwent successful cycloaddition with maleimide-terminated fluorescence dyes and a polymeric reagent. Furthermore, a nucleotide carrying a peptide via a Diels-Alder cyclohexene linkage was prepared and sequence-specifically incorporated into DNA. The Diels-Alder reaction presents a number of positive attributes such as good chemoselectivity, water compatibility, high-yield under mild conditions and no additional reagents apart from a diene and a dienophile. Furthermore, suitable dienophiles are commercially available in the form of maleimide-derivatives of fluorescent dyes and bioaffinity tags. Based on these advantages, diene- and cyclohexene-based nucleotide triphosphates are expected to find wider use in the area of nucleic acid chemistry.
Borsenberger
Vinciane
V
Department of Chemistry, Institute of Structural Molecular Biology, University College London, London WC1H 0AJ, UK.
Howorka
Stefan
S
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction
C539831:5-(pent-4-ynoic acid hexa-2,4-dienyl ester)-2'-deoxyuridine-5'-triphosphate; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D008301:Maleimides; D010455:Peptides; D004247:DNA