1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

Abstract

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono-and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5dioxohexyl group (HDO) efficiently reacted with argininecontaining peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

“…The DPA-modified dUTP was produced through nucleic acid click chemistry and this functionalised dU DPA TP was then enzymatically incorporated into the TFO sequence by SNI-PEX. ( 52 ) Although the inclusion of modified nucleotides at a specific sequence position has previously been reported, ( 54 , 56 ) the incorporation of a modification of this size had not been achieved. Additionally, to our knowledge, this is the first report of the enzymatic synthesis of an AMN-TFO hybrid.…”

“…( 53 ) However, it was not until 2013 when the Hocek group reported the SNI-PEX protocol ( 52 ) that the enzymatic synthesis of AMN-TFO hybrids became a possibility. Recently, the PEX synthesis of oligonucleotides bearing crosslinking agents ( 54 ), redox labels ( 55 ) and fluorophores ( 56 ) have been reported, indicating the expanding possibilities of this technology. In the current work, the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction ( 57 , 58 ) was employed to generate novel DPA-modified nucleotide triphosphates, which were incorporated into the TFO sequence using a DNA polymerase and employed as gene-editing tools without purification steps.…”

Formamidine-Induced Translocative Rearrangement of 2-Pyrones to 2-Pyridone Analogues with a 1,5-Diketo Motif

“…4 Recently, diketo-modified analogs showed crosslinking potential with the target protein. 5 In addition, it has the potential to act as a biomimetic ligand for metal-mediated 6 biologics. These biofunctional properties of diketo-system and 2-pyridones attract researchers to explore their synthetic route and diversification.…”