Probing the Role of the Linker in Ferrocene–ATP Conjugates: Monitoring Protein Kinase Catalyzed Phosphorylations Electrochemically

Sanela Martić, Mahmoud Labib, Daniel Freeman, Porf Heinz Bernhard Kraatz

Research output: Contribution to journalArticlepeer-review

Abstract

<jats:title>Abstract</jats:title><jats:p>The synthesis and electrochemical properties of ferrocene conjugates are presented for the purpose of investigating adenosine 5′‐[γ‐ferrocenoylalkyl] triphosphate (<jats:bold>1 a</jats:bold>–<jats:bold>4 a</jats:bold>, ferrocene (Fc)–ATP) as co‐substrates for phosphorylation reactions. Compounds <jats:bold>1 a</jats:bold>–<jats:bold>4 a</jats:bold> were synthesized, purified by HPLC, and characterized by NMR spectroscopy and mass spectrometry. In solution, all Fc–ATP bioconjugates exhibit a reversible one‐electron redox process with a half‐wave potential (<jats:italic>E</jats:italic><jats:sub>1/2</jats:sub>) in the 390–430 mV range, peak separations (Δ<jats:italic>E</jats:italic><jats:sub>p</jats:sub>) in the 40–70 mV range, and the peak current ratio (<jats:italic>i</jats:italic><jats:sub>pa</jats:sub>/<jats:italic>i</jats:italic><jats:sub>pc</jats:sub>) near unity. The peptide‐modified surface Glu‐Gly‐Ile‐Tyr‐Asp‐Val‐Pro was used to study the sarcoma‐related protein (Src) kinase activity by employing the Fc–ATP bioconjugates as co‐substrates. Subsequent kinase‐catalyzed transfer of the γ‐Fc‐phosphate group to the tyrosine residues of the surface‐bound peptides was characterized by a formal potential (<jats:italic>E</jats:italic><jats:sup>o</jats:sup>) ≈390 mV (vs. Ag/AgCl). The Fc‐coverage, estimated by time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS) and cyclic voltammetry (CV), suggested validity of Fc–ATP conjugates as kinase co‐substrates. Depending on the length of the alkyl spacer of the Fc–ATP conjugate, different current densities were obtained, pointing to a direct correlation between the two. Molecular modeling revealed that the structural constraint imposed by the short alkyl spacer (<jats:bold>1 a</jats:bold>) causes a steric congestion and negatively affects the outcome of phosphorylation reaction. An optimal analytical response was obtained with the Fc–ATP conjugates with linker lengths longer than six CH<jats:sub>2</jats:sub> groups.</jats:p>
Original languageEnglish
Pages (from-to)6744-6752
Number of pages0
JournalChemistry – A European Journal
Volume17
Issue number24
Early online date3 May 2011
DOIs
Publication statusPublished - 6 Jun 2011

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