Protein thioacylation: 2. Reagent stability in aqueous media and thioacylation kinetics.

Several thioacylating reagents have been tested toward hydrolysis under conditions suitable for protein modifications: 20-35 degrees C and buffered solutions at pH 7.5-8.5. Aliphatic dithioesters are sufficiently stable in aqueous media at room temperature (or below) if protein modification reaction time does not exceed 24 h, whereas at 35 degrees C reaction times must be limited to a few hours. Kinetic data obtained in gelatin thioacylation at room temperature using aliphatic dithioesters and dithio acid are consistent with a second-order reaction rate with respect to amine concentration. The pH dependence of the second-order reaction rate constants indicate that dithioester reacts exclusively with the free amine form of lysine residue, whereas dithiocarboxylate ion reacts with both amine and ammonium ion, probably through a more complex mechanism. Interestingly thioacylation using dithio acids may be obtained in pH near neutrality or in slightly acidic media, thus offering protein modification possibilities at pH 5-9. Thioacylation reaction rates may be expressed as R = -(dAt/dt) = k[H3O+](-b)At2[thioacylating agent] in which At is the amine concentration at time t, constants k and b depending on the reagent nature.

Protein thioacylation. 1. Reagents design and synthesis.

Thioacylation is a new way for protein chemical modification. Carboxylic dithioesters and -acids react selectively and rapidly at room temperature with aliphatic amines such as lysine epsilon-amino groups leading to thioamide formation, without any other reagent or catalyst. Various thioacylating reagents were synthesized: monofunctional dithioesters bearing on the acylating end various chemical groups such as: aliphatic chains, phenyl group, mono- and dicarboxylic acids, dialkylphosphonic ester, phosphonic acid, thiol, phenol, or quaternary ammonium group. Bifunctional dithioesters containing either a polymethylene chain or an ethylene oxide oligomer as spacer group as well as some mono- and bis(dithio acids) are described. Applications of thioacylation may be involved either in enzyme chemical modifications or in the obtention of new materials from proteins. Bifunctional reagents might be used as cross-linking or coupling reagents.