Probing the role of the linker in ferrocene-ATP conjugates: monitoring protein kinase catalyzed phosphorylations electrochemically.

Abstract

The synthesis and electrochemical properties of ferrocene conjugates are presented for the purpose of investigating adenosine 5'-[γ-ferrocenoylalkyl] triphosphate (1 a-4 a, ferrocene (Fc)-ATP) as co-substrates for phosphorylation reactions. Compounds 1 a-4 a 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 (E(1/2)) in the 390-430 mV range, peak separations (ΔE(p)) in the 40-70 mV range, and the peak current ratio (i(pa)/i(pc)) 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 (E°) ≈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 (1 a) 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(2) groups.

• Authors: Sanela Martić, Mahmoud Labib, Daniel Freeman, Porf Heinz-Bernhard Kraatz
• Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (Chemistry) Vol. 17 Issue 24 Pg. 6744-52 (Jun 06 2011) ISSN: 1521-3765 [Electronic] Germany
• PMID: 21542035 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

• Ferrous Compounds
• Metallocenes
• Peptides
• Adenosine Triphosphate
• Protein Kinases
• ferrocene

Topics

• Adenosine Triphosphate (chemistry)
• Biosensing Techniques
• Catalysis
• Electrochemistry (methods)
• Ferrous Compounds (chemical synthesis, chemistry)
• Magnetic Resonance Spectroscopy
• Mass Spectrometry
• Metallocenes
• Models, Molecular
• Molecular Structure
• Peptides (chemistry)
• Phosphorylation
• Protein Kinases (chemistry)