Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol-Ene Click Chemistry

Thiol-ene click chemistry can be exploited for the immobilization of cysteine-tagged dehydrogenases in an active form onto carbon electrodes (glassy carbon and carbon felt). The electrode surfaces have been first modified with vinylphenyl groups by electrochemical reduction of the corresponding diazonium salts generated in situ from 4vinylaniline. The grafting process has been optimized in order to not hinder the electrochemical regeneration of NAD+/ NADH cofactor and soluble mediators such as ferrocenedimethanol and [Cp*Rh(bpy)Cl](+). Having demonstrated the feasibility of thiol ene click chemistry for attaching ferrocene moieties onto those carbon surfaces, the same approach was then applied to the immobilization of D-sorbitol dehydrogenases with cysteine tag. These proteins can be effectively immobilized (as pointed out by XPS), and the cysteine tag (either 1 or 2 cysteine moieties at the N terminus of the polypeptide chain) was proven to maintain the enzymatic activity of the dehydrogenase upon grafting. The bioelectrode was applied to electroenzymatic enantioselective reduction of D -fructose to Dsorbitol, as a case study.