title: 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.affiliation: Chemistry Department, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Sanela
authors.initials: S
authors.lastname: Martić
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Labib
authors.forename: Daniel
authors.initials: D
authors.lastname: Freeman
authors.forename: Porf Heinz-Bernhard
authors.initials: PH
authors.lastname: Kraatz
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D015374:Biosensing Techniques; D002384:Catalysis; D004563:Electrochemistry / Q000379:methods*; D005296:Ferrous Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D000075163:Metallocenes; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*
chemical_list: D005296:Ferrous Compounds; D000075163:Metallocenes; D010455:Peptides; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C004998:ferrocene