Ferrocene (Fc) and its derivatives are attractive electrochemical probes for nucleic acid analysis because of their stability and convenient synthetic chemistry. Early examples of Fc labelling have utilised the conjugation of carboxy-Fc to 5'-amino-modified oligos.⁽¹⁾ Internal post-synthetic labelling of DNA probes has been obtained by reaction with ferrocenecarboxaldehyde or aminoferrocene.⁽²⁾ For direct incorporation into oligonucleotides, Fc phosphoramidites⁽³⁾ and monomers with a ferrocenyl moiety linked to position 5 of 2'- dU⁽⁴⁾ and dC or the 2’ sugar position of dA and dC⁽⁵⁾ have been described, as has on column derivatisation of I-dU with ferrocenyl propargylamide.⁽⁶⁾ Methods using redox tagging have also been employed.⁽⁷⁾

Brisset and co-workers have described the synthesis and use of abasic Fc-modified phosphoramidites,⁽⁸⁾ including the preparation of Fc-modified phosphorothioates⁽⁹⁾ however, to our knowledge, these are not commercially available. In any case, reported coupling efficiencies and oligo synthesis yields are relatively low.

To provide a robust phosphoramidite for direct incorporation into oligos we chose a structure analogous to our other dT products (eg. amino, dabcyl, biotin, fluorescein etc). This both simplifies its synthesis and imparts the benefits of having a nucleobasic structure consistent with natural DNA-sugar-phosphate backbone. Further, as the Fc-modification is on the 5-position of the pyrimidine, natural base-pairing to dA will still occur.

Ref:

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