pH Optimization of Amidation via Carbodiimides

Abstract

The use of carbodiimides to create an amine-reactive reagent is a favored means of modifying proteins, nucleic acids, and small-molecule organic compounds containing carboxylic acid groups, or vice versa. The rules for optimizing the amidation have not previously been presented quantitatively, but such optimization is critical when modifying with an expensive fluorescent dye. In this study, the reaction conditions for attaching an amine-containing dye to sodium carboxymethyl cellulose (NaCMC) via 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) are systematically examined, with an emphasis on the role played by the pKa of the coupling amine group. The reactivity is reasoned to be proportional to the product of the fractional deprotonation of NaCMC, the fractional protonation of the carbodiimide, the fractional deprotonation of the amine-containing dye, and the relative stability of the reactants and intermediates: (αCMC-)(1 – αEDAC)(αamine)Sf. The pH dependence of this product for a given amine group pKa can be used, by comparison to experimental data, to determine the pH dependence of Sf. Substitution of the pKa for a second amine-containing dye and inverting the above procedure gives a semiquantitative prediction of the pH conditions to optimize its amidation. The results suggest that generally available pKa values and the above form of Sf can be used to optimize carbodiimide assisted amidation.