Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA

Three sets of 7‐deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dN(FcX)TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox‐labelled DNA probes. Square‐wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc‐labelled A (dA(Fc)) and FcPa‐labelled C (dC(FcPa)) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox‐labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dA(Fc) and dC(FcPa) bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.