EDC is the most popular carbodiimide used for conjugating carboxylic acid to amines. It is a water soluble reagent. Both the reagent and the isourea are formed as by products during the coupling reaction and both are water soluble and may be removed easily by dialysis or gel filtration. The reagent is, however, labile in the presence of water. The chemical should be stored dessicated at -20oC. It is recommended to warm the bottle to room temperature before opening to prevent condensation occurring that will cause decomposition of the reagent over time. A concentrated solution of EDC in water may be prepared to facilitate the addition of a small molar amount to a reaction, but the stock solution should be used immediately to prevent extensive loss of activity. In aqueous solutions, hydrolysis by water is the major competing reaction, cleaving off the activated ester intermediately, forming an isourea, and regenerating the carboxylate group. For proteins the EDC mediated amide bond formation effectively occurs between pH 4.5 and 7.5. Beyond this pH range, the coupling reaction occurs more slowly with lower yields. Avoid amine and carboxylate containing buffer that may react with the carbodiimide.
Sulfo NHS esters are hydrophilic reactive groups that couple rapidly with amines on target molecules. Unlike non sulfonated NHS esters that are relatively water insoluble and must first be dissolved in organic solvent before adding to aqueous solutions, sulfo NHS esters typically are water soluble and don’t hydrolyze as quickly in water. However, in the presence of amine nucleophiles that can attack at the carbonyl group of the ester, the sulfo NHS group readily leaves, creating a stable amide linkage with the amine. Sulfhydryl and hydroxyl groups will also react with active esters, but the products of these reactions, thioesters and eaters, are relatively unstable compared to an amide bond.
The advantage of adding sulfo NHS to EDC reactions is to increase the solubility and stability of the active intermediate, which ultimately reacts with the amine. The active ester formed by EDC is very slow to react with amines and can hydrolyze in aqueous solutions. If the target amine does not find the active carboxylate before it hydrolyzes, the desired coupling cannot occur. Hence, forming a sulfo NHS ester intermediate increases the resultant amide bond formation. Since the concentration of added sulfo NHS is greater than the concentration of the target molecule, the reaction preferentially proceeds through more the efficient sulfo NHS ester intermediate. The combination of EDC-sulfo NHS results in significantly higher coupling efficiency and increases in yield of conjugation over that obtainable solely from EDC alone.