pH Optimization of Amidation via Carbodiimides

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 pK(a) 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: (alpha(CMC))(1 - alpha(EDAC))(alpha(amine))S-f. 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 S-f. Substitution of the pK(a) 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 pK(a) values and the above form of S-f can be used to optimize carbodiimide assisted amidation.