Kinetics and mechanism of the reversible isomerization of aspartic acid residues in tetrapeptides

Abstract

The influence of pH and buffer concentration on the rate of the isomerization of Asp residues has been analysed using model aspartic acid-containing tetrapeptides, in the pH range 1.5-1 0 at 37 "C and p = 1 mol dm-3. The reaction involves the reversible formation of an amino-succinimide intermediate.Kinetic evidence indicates that of the various forms of the Asp-peptide in acid-base equilibrium, only one, having the carboxylic Asp side chain in the neutral state and the N-H peptide group next to the Asp residue in the deprotonated state, reacts at an appreciable rate. The reaction involves nucleophilic attack b y the nitrogen atom of the peptide bond o n the carbonyl carbon of the Asp side chain, giving a cyclic tetrahedral intermediate. A t p H values lower than about 3 the cyclization step is rate-determining, but at higher pH the rate-determining step is the general acid-catalysed departure of the leaving group. This change of rate-determining step, together with the ionization of the carboxylic side chain of the Asp residue, produces a bell-shaped curve in the pH-rate profile for the cyclization reactions.

Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures

“…In mildly acidic solutions, the rate of cyclic imide formation is higher than the rate of its hydrolysis. Consequently the succinimide intermediate can accumulate (11)(12)(13).…”

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

“…1,2 This has been attributed to the formation of aminosuccinimidyl (Asu) peptides (Scheme 1) arising from the intramolecular nucleophilic attack of the side chain CO group by the a-nitrogen of the peptide backbone amide. 3,4 The succinimide can subsequently hydrolyze to yield the respective aspartyl (Asp or a-Asp) peptides and iso-aspartyl (iso-Asp or b-Asp) peptides favoring the b-Asp peptide over the ''normal'' a-Asp linkage at a ratio between 5:1 and 3:1. 5,6 Formation of the succinimide and subsequent isomerization largely depend on steric hindrance and conformational space.…”

Formulation Development for Antibody-Drug Conjugates

“…A faster rate of attack by nitrogen at higher pH is canceled out because O−, formed readily at high pH, is a poor leaving group. As a result, the ring closure reaction of Asp is pH independent from pH 5 or above (Capasso et al 1995). Factors other than pH such as sequence and steric effect of the side chain of neighboring amino acids may also influences the rate of deamidation and isomerization in proteins (Tyler-Cross and Schirch 1991;Son and Kwon 1995).…”

“…(Data reconstructed from Gieger and Clarke (1987), Capasso et al (1993Capasso et al ( , 1995 independent of succinimide formation (Goswami et al 2013). While the rate of deamidation increases rapidly with the increase of pH, the pH-rate profiles of Asp show its pH dependency in the range of pH 3-6 and then no effect above pH 7 with a sharp maximum at a pH close to the apparent pKa of the corresponding carboxyl group.…”

“…The pH-rate profile of deamidation, isomerization, and succinimide hydrolysis was established using a model tetrapeptide, as shown in Fig. 5.5 (Gieger and Clarke 1987;Capasso et al 1993Capasso et al , 1995, which illustrates the dependency of the reactions on hydroxide ion concentration. Similar observation can be found in the thio-succinimide reaction.…”