Peptide fragmentation can lead to an oxazolone or diketopiperazine b2+ ion structure. IRMPD spectroscopy combined with computational modeling and gas-phase H/D exchange was used to study the structure of the b2+ ion from protonated HAAAA. The experimental spectrum of the b2+ ion matches both the experimental spectrum for the protonated cyclic dipeptide HA (a commercial diketopiperazine) and the theoretical spectrum for a diketopiperazine protonated at the imidazole pi nitrogen. A characteristic band at 1875 cm−1, and increased abundance of the peaks at 1619 and 1683 cm−1, indicate a second population corresponding to an oxazolone species. H/D exchange also shows a mixture of two populations consistent with a mixture of b2+ ion diketopiperazine and oxazolone structures.

Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b2+ ion structures have been of recent interest. While previously studied b2+ ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b2+ ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analogue-containing Xxx-Ala b2+ ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen-deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b2+ ion structures formed. The b2+ ions studied include His-Ala (HA), methylated histidine analogues, including pi-methyl-HA and tau-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid (DabA) and Lys-Ala (KA). The location and accessibility of the histidine pi nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the tau nitrogen did not change the b2+ ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans-cis isomerization of the first amide bond that is necessary for diketopiperazine formation.