Mass spectral and theoretical investigations of the transient proton-bound dimers on the cleavage processes of the peptide GHK and its analogues

Fragmentation mechanisms of the singly protonated peptides GHK, GHKH and HGHK have been investigated by mass spectrometry and theoretical calculations. Fragmentation behavior of the protonated H–K amide bond in GHK was changed completely when a histidinyl residue was introduced into the C-terminus of GHK. The H–K amide bond breaking was a predominant pathway in the case of GHK and GHKH. For HGHK, the histidinyl residue at the N-terminus hampered significantly breaking of the H–K amide bond resulting in a high potential energy barrier; calculations indicated that this histidinyl effect played a vital role for the H–K amide bond fragmentation. Subsequent analysis of the fragmentation mechanism revealed that recombination processes of the hydrogen bonding for the intermediate products were all exergonic. Formation of a proton-bound dimer (PBD) lowering the energy barriers from a thermodynamic perspective for all the designed fragmentation pathways was demonstrated to be feasible by our systematic calculations. Moreover, the involvement of different PBDs was further confirmed by analyses of the reduced density gradient (RDG) isosurfaces and scatter maps. A dynamically favored pathway was likely via six-membered ring or nine-membered ring structures generated by the diketopiperazine as revealed by atom-in-molecules (AIM) analyses, since the steric interaction energies in the newly formed ring were estimated to be relatively small when compared to the products generated from a lactam and/or an oxazolone pathway. This is the first feasibility investigation from a dynamic viewpoint for formation of different rings involved in the lactam, oxazolone or diketopiperazine pathways in the fragmentation mechanisms proposed.