Indigenous functional groups of an antibody such as carboxylic acid can be activated using suitable coupling reagents and the resulting reactive intermediate can be used for conjugation to lipids containing amine groups. This results in the formation of a stable amide linkage.
N-substituted carbodiimides can react with carboxylic acids to form a highly reactive O-acylurea derivatives that are extremely short lived. This active species can react with nucleophile such as primary amine to form amide bond. Other nucleophiles also are reactive such as sulfhydryl groups may attack and form thioester linkages, although they are not as stable as the bond formed with an amine. In addition, oxygen atoms may act as nucleophile such as those in water molecules. In an aqueous solution, hydrolysis by water is the major competing reaction, both inactivating EDC itself and cleaving off the activated ester intermediate, forming an isourea, and regenerating the carboxylate group. Furthermore hyrazide containing compounds can also be coupled to the activated carboxylate group thus modifying the carboxylate to possess terminal hydrazide groups which then can be used for conjugation to other carbonyl compounds. EDC mediated amide bond formation effectively occurs between pH 4.5 and 7.5