Diketopiperazine Formation from FPGnK (n = 1-9) Peptides: Rates of Structural Rearrangements and Mechanisms.

Peptides with penultimate proline residues undergo trans → cis isomerization of the Phe1-Pro2 peptide bond followed by spontaneous bond cleavage at the Pro2-Xxx3 bond (where Xxx is another amino acid residue), leading to cleavage of the Pro2-Xxx3 bond and formation of a diketopiperazine (DKP). In this paper, ion mobility spectrometry and mass spectrometry techniques were used to study the dissociation kinetics of nine peptides [Phe1-Pro2-Glyn-Lysn+3 (n = 1-9)] in ethanol. Shorter (n = 1-3) peptides are found to be more stable than longer (n = 4-9) peptides. Alanine substitution studies indicate that, when experiments are initiated, the Phe1-Pro2 bond of the n = 9 peptide exists exclusively in the cis configuration, while the n = 1-8 peptides appear to exist initially with both cis- and trans-Phe1-Pro2 configured bonds. Molecular dynamics simulations indicate that intramolecular hydrogen bonding interactions stabilize conformations of shorter peptides, thus inhibiting DKP formation. Similar stabilizing interactions appear less frequently in longer peptides. In addition, in smaller peptides, the N-terminal amino group is more likely to be charged compared to the same group in longer peptides, which would inhibit the dissociation through the DKP formation mechanism. Analysis of temperature-dependent kinetics measurements provides insight about the mechanism of bond cleavage. The analysis gives the following transition state thermochemistry: ΔG⧧ values range from 94.6 ± 0.9 to 101.5 ± 1.9 kJ·mol-1, values of ΔH⧧ range from 89.1 ± 0.9 to 116.7 ± 1.5 kJ·mol-1, and ΔS⧧ values range from -25.4 ± 2.6 to 50.8 ± 4.2 J·mol-1·K-1. Proposed mechanisms and thermochemistry are discussed.