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.
Highlights
Deamidation of asparagine (Asn, N) residues is one of the most common reactions which occur nonenzymatically in peptide chains
A succinimide species is known to be the intermediate of Asn deamidation (Scheme 1) [1–11]. This intermediate having a five-membered ring is formed by the nucleophilic attack of the main-chain nitrogen atom of the C-terminal adjacent residue on the Asn side-chain amide carbon with release of an ammonia molecule
The nucleophilic attack gives rise to a five-membered ring tetrahedral intermediate
Summary
Deamidation of asparagine (Asn, N) residues is one of the most common reactions which occur nonenzymatically in peptide chains. A succinimide species is known to be the intermediate of Asn deamidation (Scheme 1) [1–11]. This intermediate having a five-membered ring is formed by the nucleophilic attack of the main-chain nitrogen atom of the C-terminal adjacent residue on the Asn side-chain amide carbon with release of an ammonia molecule. This is an intramolecular nucleophilic substitution reaction and is generally considered to occur in two steps (cyclization-deammoniation, Scheme 2) [12,13]. While the Asn deamidation in proteins and peptides is often regarded as a degradation reaction, a “molecular clock” hypothesis was
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