Modeling the Enolization of Succinimide Derivatives, a Key Step of Racemization of Aspartic Acid Residues: Importance of a Two‐H2O Mechanism

Racemization of aspartic acid residues in peptides and proteins is assumed to proceed via succinimide intermediates. An enolization of the succinimide intermediate is required for the racemization to occur. In this study, we modeled the enolization step by density‐functional theory (DFT) calculations (B3LYP/6‐31+G**), using two model compounds, N‐methylsuccinimide (1) and its formylamino derivarive 2. Three mechanisms were investigated for 1, i.e., the direct mechanism without active participation of H2O molecules, and one‐H2O and two‐H2O mechanisms, in which one or two H2O molecules actively participate in the reaction. We found that the two‐H2O mechanism was the most favorable with an activation barrier of 37 kcal mol−1. In the two‐H2O mechanism, a concerted bond reorganization involving a triple H‐atom transfer occurred in an eight‐membered cyclic structure formed between the imide and two H2O molecules. For 2, we investigated only the two‐H2O mechanism and found that the activation barrier was lowered to 31 kcal mol−1 due to an H‐bond between the CO O‐atom of the formylamino group (‘the neighboring residue’) and one of the H2O molecules. Our results suggest that, in proteins, the Asp racemization is severely controlled by the accessibility of H2O molecules to the reaction site of the succinimide intermediate.