Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study

Abstract

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.

Scheme 1

Nonenzymatic reactions of aspartic acid (Asp) residues via the succinimide intermediate (aminosuccinyl (Asu) residue).

Scheme 2

Two-step mechanism for succinimide formation from an Asp residue.

Figure 1

The model compound used in the present study (Ace-Asp-Nme). The φ (C–N–C_α–C) and ψ (N–C_α–C–N) dihedral angles, which characterize the main-chain conformation, and the χ₁ dihedral angle (N–C_α–C_β–C_γ), which characterizes the side-chain conformation, are indicated.

Figure 2

Energy diagram (kcal·mol⁻¹), where relative energies corrected for the zero-point energy (ZPE) and the SM8 (solvation model 8) hydration free energy are shown with respected to the reactant complex, R•AA (R, reactant molecule; AA, acetic acid). The ZPE-corrected relative energies in a vacuum are shown in parentheses for comparison. The single imaginary frequency (cm⁻¹) is also shown for TS-1 and TS-2 (TS, transition state). I, intermediate; P, product molecule; W, water.

Figure 3

The geometries of (a) the reactant R (model compound, Figure 1) (φ = −162°, ψ = 162°, χ₁ = 72°) and (b) the succinimide product P (φ = −171°, ψ = −141°, χ₁ = 136°).

Figure 4

The geometry of the reactant complex R•AA (φ = −164°, ψ = −178°, χ₁ = 76°). The α carbon atom is indicated by an asterisk (*). Selected interatomic distances are shown in Å. The gas-phase total energy of this geometry is −913.600369 E_h.

Figure 5

The geometry of the transition state TS-1 of the first step (cyclization) (φ = −162°, ψ = −150°, χ₁ = 115°). The distances of forming and breaking bonds are shown in Å. The asterisk (*) indicates the α carbon.

Figure 6

The geometry of I•AA-1 (φ = −167°, ψ = −138°, χ₁ = 117°), which is the intermediate complex directly connected to TS-1. Selected interatomic distances are shown in Å. The asterisk (*) indicates the α carbon.

Figure 7

The geometry of I•AA-2 (φ = −170°, ψ = −145°, χ₁ = 146°), which is the intermediate complex directly connected to TS-2. Selected interatomic distances are shown in Å. The asterisk (*) indicates the α carbon.

Figure 8

The geometry of the transition state TS-2 of the second step (dehydration) (φ = −169°, ψ = −142°, χ₁ = 144°). The distances of forming and breaking bonds are shown in Å. The asterisk (*) indicates the α carbon.

Figure 9

The geometry of the product complex P•AA•W (φ = −173°, ψ = −140°, χ₁ = 137°) formed by the succinimide product P, the regenerated acetic acid molecule AA and the released water molecule W. Selected interatomic distances are shown in Å. The asterisk (*) indicates the α carbon.