Influence of N Terminus Amino Acid on Peptide Cleavage in Solution through Diketopiperazine Formation.

Abstract

Diketopiperazine (DKP) formation is an important degradation pathway for peptides and proteins. It can occur during synthesis and storage in either solution or the solid state. The kinetics of peptide cleavage through DKP formation have been analyzed for the model peptides Xaa1-Pro2-Gly4-Lys7 [Xaa = Gln, Glu, Lys, Ser, Phe, Trp, Tyr, Cha (β-cyclohexylalanine), Aib (α-aminoisobutyric acid), Gly, and Val] at multiple elevated temperatures in ethanol with ion mobility spectrometry-mass spectrometry (IMS-MS). When Xaa is an amino acid with a charged or polar side chain, degradation is relatively fast. When Xaa is an amino acid with a nonpolar alkyl side chain, the peptide is relatively stable. For these peptides, a bulky group on the α carbon speeds up dissociation, but the kinetic effects vary in a complicated manner for bulky groups on the β or γ carbon. Peptides where Xaa has a nonpolar aromatic side chain show moderate dissociation rates. The stability of these peptides is a result of multiple factors. The reaction rate is enhanced by (1) the stabilization of the late transition state through the interaction of an aromatic ring with the nascent DKP ring or lowering the activation energy of nucleophilic attack intermediate state through polar or charged residues and (2) the preference of the cis proline bond favored by the aromatic N-terminus. The number of unseen intermediates and transition state thermodynamic values are derived for each peptide by modeling the kinetics data. Most of the transition states are entropically favored (ΔS⧧ ∼ -5 to +31 J·mol-1·K-1), and all are enthalpically disfavored (ΔH⧧ ∼ 93 to 109 kJ·mol-1). The Gibbs free energy of activation is similar for all of the peptides studied here (ΔG⧧ ∼ 90-99 kJ·mol-1).