Key featuresShow Hide
- Useful as electrochemical probe for nucleic acid analysis.
- Allows stable direct incorporation into oligos with nucleobasic structure consistent with natural DNA-sugar-phosphate backbone.
- As the Fc-modification is on the 5-position of the pyrimidine, natural base-pairing to dA will still occur.
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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.
- (a) Electrochemically active DNA probes: Detection of target DNA sequences at femtomole level by high-performance liquid chromatography with electrochemical detection, S. Takenaka, Y. Uto, H. Kondo, T. Ihara and M. Takagi, Anal. Biochem., 218, 436-443, 1994; (b) Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA, T. Ihara, Y. Maruo, S. Takenaka and M. Takagi, Nucleic Acids Research, 24, 4273-4280, 1996; (c) Electrochemical analysis of DNA amplified by the polymerase chain reaction with a ferrocenylated oligonucleotide, Y. Uto, H. Kondo, M. Abe, T. Suzuki and S. Takenaka, Anal. Biochem., 250, 122-124, 1997.
- Electrochemical detection of sequence-specific DNA using a DNA probe labelled with aminoferrocene and chitosan modified electrode immobilized with ssDNA, C. Xu, H. Cai, P. He and Y. Fang, Analyst, 126, 62-65, 2001.
- (a) M. Wiessler and D. Schutte, European Patent WO9709337 (1997); (b) T.Chunlin, US Patent Application US2009/0155795 A1 (2009).
- (a) Uridine-conjugated ferrocene DNA oligonucleotides: Unexpected cyclization reaction of the uridine base, C.J. Yu, H. Yowanto, Y. Wan, T.J. Meade, Y. Chong, M. Strong, L.H. Donilon, J.F. Kayyem, M. Gozin and G.F. Blackburn, J. Amer, Chem. Soc., 122, 6767-6768, 2000; (b) Ferrocene-modified pyrimidine nucleosides: synthesis, structure and electrochemistry, H. Song, X. Li, Y. Long, G. Schatte and H.-B. Kraatz, Dalton Trans., 4696-4701, 2006.
- 2'-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids, C.J. Yu, H. Wang, Y. Wan, H. Yowanto, J.C. Kim, L.H. Donilon, C. Tao, M. Strong and Y. Chong, J. Org. Chem., 66, 2937-2942, 2001.
- On-column derivatization of oligodeoxynucleotides with ferrocene, A.E. Beilstein and M.W. Grinstaff, Chem. Commun., 509-510, 2000.
- (a) Synthesis of the first ferrocene-labelled dideoxynucleotide and its use for the 3'-redox end-labelling of 5'-modified single-stranded oligonucleotides, A. Anne, B. Blanc and J. Moiroux, Bioconjug. Chem., 12, 396-405, 2001; (b) Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA, W.A Wlassoff and G.C. King, Nucleic Acids Research, 30, e58, 2002.
- Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution, A.E. Navarro, N. Spinelli, C. Moustrou, C. Chaix, B. Mandrand and H. Brisset, Nucleic Acids Research, 32, 5310- 5319, 2004; (b) Supported synthesis of ferrocene modified oligonucleotides as new electroactive DNA probes, A.-E. Navarro, N. Spinelli, C. Chaix, C. Moustrou, B. Mandrand and H. Brisset, Bioorg. Med. Chem. Lett., 14, 2439-2441, 2004; (c) C. Chaix-Bauvais et al, US Patent Application US2005/0038234 A1 (2005).
- The first automated synthesis of ferrocene-labelled phosphorothioate DNA probe: A new potential tool for the fabrication of microarrays, H. Brisset, A.-E. Navarro, N. Spinelli, C. Chaix and B. Mandrand, Biotechnol. J., 1, 95-98, 2006.