Free Radical-Mediated Selective C-C Bond Cleavage and Differentiation of Isomers in Peptide Derivatives

The selective C-C bond activation is a frontier research topic and of great significance in the field of chemistry, especially for biosciences. Due to the existence of many C-C bonds with similar activities in compounds, it is difficult to selectively activate one of the C-C bonds. In this paper, the newly synthesized peptide derivatives composed of unnatural amino acids were used as a subject to demonstrate how radical-mediated selective C-C bond activation and isomer differentiation work. TEMPO radical initiator was employed to introduce o-methylbenzoyl (Bz) radical into the peptide derivatives, and successfully prepared[Bz-M+H]•+radical ions in the gas phase. Through tandem mass spectrometry experiments, it had been found that[Bz-M+H]•+ showed higher reactivity than the protonated peptide molecule[M+H]+, giving a more diversified gas-phase dissociation reactions. The main fragmentation of[M+H]+ was the cleavage of amide bond to give rise to[y1+2H]+ (m/z 160.134 0, RA 100%), a1+ (m/z 86.097 2, RA 74%), and[(M+H)-HCOOEt]+ (m/z 199.181 5, RA 52%). In contrast, the fragment ions of[Bz-M+H]•+ included[Bz•-a1]+ (m/z 202.97),[Bz•-b1]+ (m/z 231.03),[Bz•-c1+H]+ (m/z 133.95) and[(Bz-M+H)-HCOOEt]+ (m/z 316.18, RA 100%). N-terminal fragments of[Bz-M+H]•+ were observed with the radical part •CH2C6H4CO still attached to the fragmentation. To distinguish these ions from the normal fragments, the prefix Bz• was introduced, such as,[Bz•-a1]+. More interestingly,[(Bz-M+H)-HCOOEt]+ was the base peak of[Bz-M+H]•+, which was produced by breaking the Cα-C adjacent to the ester group of peptide derivative. In contrast, the relative abundance of[(M+H)-HCOOEt]+ from[M+H]+ was only 50%. The formation mechanism of[(M+H)-HCOOEt]+ had been experimentally confirmed to be completed by the two-step reaction of losing EtOH and CO successively. For isomers B1 and B2, their CID spectra of[Bz-M+H]•+ had high similarity, but the abundance of fragment ion[Bz•-a1]+ was obviously different (m/z 203, RA 40% for B1, RA 60% for B2). The fragment ions[Bz•-a1]+ and[(Bz-M+H)-HCOOEt]+ of[Bz-M+H]•+ can be used as sensitive probes for isomer discrimination and selective C-C bond cleavage. The research provides new strategy to distinguish peptide isomers and to cleavage selective C-C bond with radical participation in mass spectrometer. © 2022, Editorial Board of Journal of Chinese Mass Spectrometry Society. All right reserved.