However, this intermediate also can

However, this intermediate also can undergo a hydrolysis event to yield the same undesired isourea derivative, which again inactivates the compound. Therefore, there are two stages in the reaction sequence in which hydrolysis can occur and inhibit the desired product formation (Nakajima and Ikada, 1995). If hydrolysis does not occur, there are at least three subsequent reactions that can happen, including the desired amide bond formation. If a neighboring carboxylate group is in close proximity to the O-acylisourea ester, it may react with it and form an anhydride intermediate. This especially can occur in polymers containing repeating carboxylate groups, such as in polymethacrylate, where the primary intermediate reactive group formed from EDC may be anhydrides (Wang et al., 2011). Fortunately, ananhydride is also reactive with amine groups, so the desired amide bond formation can still occur with at least one of the two carboxylates making up the anhydride. Indeed, anhydride formation may result in higher yields of amide bond formation in certain instances (Nakajima and Ikada, 1995). In addition, if EDC is in large excess over the amount of carboxylates present, then the intermediate ester may exist for a longer period and potentially it can rearrange by reacting with the neighboring secondary amines in the carbodiimide and thus form an N-acylisourea derivative, which is inactive and permanently attaches the EDC derivative to the carboxylate compound. Finally, the EDC-reactive ester can also react with the desired amine-containing molecule and form an amide bond. Given this degree of potential side reactions, it is amazing that EDC-mediated amide bond formation can be done with reproducibility, especially when scaling up reactions in production processes. The propensity for EDC to undergo side reactions may be a reason that high variability has been reported using the carbodiimide for particular conjugation reactions (Young et al., 2004).