Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain

Abstract

The Asn-Gly-Arg (NGR) motif and its deamidation product isoAsp-Gly-Arg (isoDGR) have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding isoDGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH2CO-NGRC]-NH2, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H2PO4− ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8) continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H2PO4− ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH2 group on the five-membered ring, was shown to easily undergo NH3 elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy

“…Two H 2 O molecules and one dihydrogen phosphate (H 2 PO 4 − ) ion or one bicarbonate (HCO 3 − ) ion was included in the calculations as catalysts. These molecules and ions abundantly exist in vivo and have been suggested to catalyze post-translational modification of proteins [ 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. The optimized geometries of the cyclization step, which occur by nucleophilic attack by the ( N + 1) amide nitrogen to carbonyl carbon of the side chain, for the syn conformation in the phosphate-catalyzed reaction are shown in Figure 8 .…”

“…Some computational studies have reported the succinimide formation mechanism of Asn and the changes of dihedral angles with the deamidation reaction using quantum chemical calculations [ 41 , 42 , 43 , 44 ]. The structural changes with succinimide formation from Asn are relatively small (change of N‒C α ‒C‒N and N‒C α ‒C β ‒C γ : 35–50°, C‒N‒C α ‒C: nearly constant) in comparison with other nonenzymatic modifications in some previous studies (the maximum change of dihedral angles: 214°) [ 45 , 46 , 47 , 48 , 49 ].…”

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

“…However, arginine has been shown to facilitate Asu formation of an adjacent NG motif at near neutral and slightly acidic pHs by coordinating buffer anions that subsequently induce Asu cyclization via proton transfer. 52 Therefore, if arginine is contributing to the observed modification, it would be highly dependent on conformation and the chemistry of the local environment.…”

In Vivo Tumor Growth Inhibition and Antiangiogenic Effect of Cyclic NGR Peptide-Daunorubicin Conjugates Developed for Targeted Drug Delivery

“…Next to daunorubicin, other drugs such as platinum IV, carboplatin and 5-fluorouracil were coupled to NGR peptides as well [43][44][45]. However, it is well known that the NGR motif can easily undergo Asn deamidation through succinimide formation followed by hydrolysis that can occur at both carbonyl groups of the cyclic imide leading to the formation of Asp and isoAsp residues with a usual ratio of 1:3 [46][47][48][49]. The speed of this process is highly influenced by numerous factors like peptide structure, temperature or pH of the solution.…”