Gas-phase studies on the reactivity of charged aromatic biradicals toward amino acids in a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer

George O Pates, Purdue University

Abstract

Biradical reaction intermediates can abstract a hydrogen atom from the sugar moiety in both strands of double-helix DNA, leading to irreversible DNA cleavage and eventually cell death. Little is known about the reaction kinetics and selectivity of aromatic carbon-centered σ,σ-biradicals. The ability of biradical intermediates to damage DNA has increased interest in their biological reactivity. Gas-phase studies of σ,σ-biradicals with dinucleoside phosphates have shown that they can abstract a base. Even further, attack of biradicals on nucleic acid components and proteins causes them to fragment, oxidize, denature, and lose enzymatic activity. The size and complexity of proteins make it difficult to obtain knowledge of these processes at the molecular level. Hence, many solution studies aimed at improving the understanding of the reactivity of biradicals towards proteins have been carried out by using free amino acids and small peptides as the substrate. A better understanding of the reactivity controlling factors involved in the reactions of biradicals can provide important information on the role of biradicals in protein damage. The reactivity of ten charged phenyl radicals toward several non-aromatic amino acids was examined in the gas phase in a dual-cell Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. All radicals abstract a hydrogen atom from the amino acids, as expected. The most electrophilic radicals also react with these amino acids via NH2 abstraction (a nonradical nucleophilic addition-elimination reaction). Both the radical (hydrogen atom abstraction) and nonradical (NH2 abstraction) reactions' efficiencies were found to increase with the electrophilicity of the radical. However, the NH2 abstraction is more strongly influenced by EA. Studies using several partially deuteriumlabeled amino acids revealed that abstraction of a hydrogen atom from the α-carbon is only preferred for glycine; for the other amino acids, a hydrogen atom is preferentially abstracted from the side chain. The electrophilicity of the radicals does not appear to have a major influence on the site from which the hydrogen atom is abstracted. Hence, the regioselectivity of hydrogen atom abstraction appears to be independent of the structure of the radical but dependent on the structure of the amino acid. Both NH2 and 15NH2 groups were abstracted from lysine labeled with 15N on the side-chain, indicating that NH2 abstraction occurs both from the amino acid backbone as well as from the side-chain. The reactivity of positively charged aromatic carbon-centered σ,σ-biradicals with varying calculated electron affinities (EA), singlet triplet (S-T) gaps and dehydrocarbon atom separation toward several amino acids was examined in the gas phase in a dual-cell Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. A distance of 2.3 Å´ was previously reported as the average didehydrocarbon atom separation for biradicals ( meta-benzynes) in the transition state for the abstraction of a hydrogen atom from methane. That energy needed for a biradical (meta-benzyne) to obtain a didehydrocarbon atom separation of 2.3 Å´ shows a correlation with the extent of both radical and nonradical reactivity. Some biradicals show increased reactivity (higher reaction efficiencies and more reaction products) towards the amino acids studied. Some of the biradicals studied here react slowly if at all with the amino acids studied and have a preference for forming a stable adduct with the amino acids via nonradical reaction pathways. Hence, both radical and nonradical reaction pathways are influenced by the dehydrcarbon atom separation. It was found that while the structure of the amino acid influences the reactivity, the structure of the biradical is a more important reactivity controlling parameter. The opposite is true for monoradicals where regioselectivity towards the amino acids appears to be independent of the structure of the radical and mostly dependent on the structure of the amino acid. Of the amino acids studied in this laboratory, proline was found to exhibit the most distinctive reactivity when allowed to react with charged monoradicals. This distinct reactivity can be attributed to proline being conformationally unique among the amino acids in that its' ϕ-torsion angle is restricted by its' characteristic five-membered pyrrolidine ring structure. Proline is the only amino acid found in mammalian proteins that contains a secondary amino group. The unique structure of proline has influence on the fragmentation of both protonated and deprotonated proline-containing peptides. In this study we demonstrate how the “proline effect” is not only observed for unimolecular fragmentation of peptides but also in radical reactions of free proline and proline containing peptides.

Degree

Ph.D.

Advisors

Kenttamaa, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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