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.