Isomerization and dissociation of aromatic-containing radical cationic peptides

Abstract

Gas phase odd-electron radical cationic peptides (M•+) display rich and diverse ion chemistry that differs substantially from that of their protonated counterparts. Dissociation of M•+ species can provide relevant and complementary structural information for protein sequencing. This thesis presents a detailed study of the isomerization and fragmentation steps in the unimolecular gas phase chemistry of phenylalanyl-, tyrosyl-, and tryptophanyl-containing M•+ species. The ability to form different types of M•+ species with various well-defined radical sites is critically important to understanding their isomerization and fragmen-tation. Chapter 1 provides an in-depth review on the ion chemistry of radical cationic peptides. Chapter 2 then gives a report on the general experimental procedures. Chapter 3 discusses four isomeric phenylalanylglycyltryptophan radical cations, with different well-defined initial radical sites, that have been synthesized experimentally in the gas phase for the first time. The π-centered [FGW]π•+ and tryptophan nitrogen-centered [FGWN•]+ species gave almost identical collision-induced dissociation (CID) spectra with common fragment ions, indicating their ready inter-convertibility. The fragmentations of the middle α-carbon ([FGα•W]+) and phenylalanine ζ-carbon ([Fζ•GW]+) radical cations were substantially different from those of [FGW]π•+ and [FGWN•]+, indicating that direct dissociations are competitive with isomerization followed by dissociation. The isomerization barriers of all three non-interconverting isomers have been examined theoretically; they are all energetically unfavorable, consistent with their substantially different reactivities. The structural, energetic, and mechanistic features of selected novel types of backbone (Chapter 4) and side-chain (Chapter 5) cleavages have been examined systematically using an integrated approach involving low-energy CID experiments and density functional theory (DFT) calculations in combination with isotopic labeling experiments. Chapter 4 describes an investigation of the dissociation of [zn – H]•+ ions from tyrosine- or tryptophan-containing peptide radical cations. Isotopic labeling experiments and DFT calculations reveal a novel nucleophilic attack by a backbone amide nitrogen atom on the side chain aromatic ring and subsequent proton transfer steps, involving unprecedented cyclization rearrangement between the peptide backbone and aromatic side chain, to produce the residue-specific products. The side chain of the tyrosyl-residue could also interact with the N-terminal amino groups to induce unusual Cα−Cβ bond cleavages of the N-terminal phenylalanine, valine, and isoleucine residues with complementary sequencing information (Chapter 5). Notably, side chain loss of phenylalanine has proven to be challenging because of the absence of a -radical. Thus, the mechanism of N-terminal phenylalanine Cα−Cβ bond cleavage (91 Da loss) has been examined systematically. Radical migration from the side chain of the tyrosine residue to the close-proximity N-terminal amino group of the phenylalanyl residue induces loss of the entire benzyl side chain, enabled by through-space electron transfer. Increase in the electron density of the N-terminal nitrogen atom of the phenyl residue facilitates the electron transfer process, leading to much more favorable neutral benzyl loss. Characterization of the non-convertible radical peptide isomers undergoing site-specific radical-mediated backbone/side chain cleavages in Chapters 4 and 5 has led to the discovery of new intermediates and products having unusual cyclic structures and displaying fascinating unimolecular gas phase chemistry.