In proteins and peptides, d-aspartic acid (d-Asp) and d-β-Asp residues can be spontaneously formed via racemization of the succinimide intermediate formed from l-Asp and l-asparagine (l-Asn) residues. These biologically uncommon amino acid residues are known to have relevance to aging and pathologies. Although nonenzymatic, the succinimide racemization will not occur without a catalyst at room or biological temperature. In the present study, we computationally investigated the mechanism of succinimide racemization catalyzed by dihydrogen phosphate ion, H₂PO₄⁻, by B3LYP/6-31+G(d,p) density functional theory calculations, using a model compound in which an aminosuccinyl (Asu) residue is capped with acetyl (Ace) and NCH₃ (Nme) groups on the N- and C-termini, respectively (Ace–Asu–Nme). It was shown that an H₂PO₄⁻ ion can catalyze the enolization of the H_α–C_α–C=O portion of the Asu residue by acting as a proton-transfer mediator. The resulting complex between the enol form and H₂PO₄⁻ corresponds to a very flat intermediate region on the potential energy surface lying between the initial reactant complex and its mirror-image geometry. The calculated activation barrier (18.8 kcal·mol⁻¹ after corrections for the zero-point energy and the Gibbs energy of hydration) for the enolization was consistent with the experimental activation energies of Asp racemization.

Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the β-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol⁻¹) agrees well with an experimental activation energy.

Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

Aspartic acid (Asp) residues in proteins and peptides are prone to the non-enzymatic reactions that give biologically uncommon l-β-Asp, d-Asp, and d-β-Asp residues via the cyclic succinimide intermediate (aminosuccinyl residue, Suc). These abnormal Asp residues are known to have relevance to aging and pathologies. Despite being non-enzymatic, the Suc formation is thought to require a catalyst under physiological conditions. In this study, we computationally investigated the mechanism of the Suc formation from Asp residues that were catalyzed by the dihydrogen phosphate ion, H₂PO₄⁻. We used Ac–l-Asp–NHMe (Ac = acetyl, NHMe = methylamino) as a model compound. The H₂PO₄⁻ ion (as a catalyst) and two explicit water molecules (as solvent molecules stabilizing the negative charge) were included in the calculations. All of the calculations were performed by density functional theory with the B3LYP functional. We revealed a phosphate-catalyzed two-step mechanism (cyclization–dehydration) of the Suc formation, where the first step is predicted to be rate-determining. In both steps, the reaction involved a proton relay mediated by the H₂PO₄⁻ ion. The calculated activation barrier for this mechanism (100.3 kJ mol⁻¹) is in reasonable agreement with an experimental activation energy (107 kJ mol⁻¹) for the Suc formation from an Asp-containing peptide in a phosphate buffer, supporting the catalytic mechanism of the H₂PO₄⁻ ion that is revealed in this study.