This dissertation, "Formation, Isomerization and Dissociation of Radical Cationic Peptides" by Chun-ming, Dominic, Ng, 伍俊明, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: A fundamental understanding of the isomerization and fragmentation of peptide ions forms the scientific basis underlying peptide sequencing in the gas phase-an important emerging analytical technique routinely used in proteomics applications. Gas phase dissociation of odd-electron radical peptide cations (M?+) provides an alternative and complementary analytical method for identifying peptide sequences; this fragmentation behavior is distinct from that of even-electron protonated peptides ([M]H]+). Despite recent experimental and theoretical advances in studies of radical cationic peptides, their gas phase chemistry remains poorly understood. The first part of this Thesis documents three mechanistic studies on the formation, isomerization, and dissociation of prototypical tripeptide radical cations in the gas phase using biological mass spectrometry. A combination of low-energy collision-induced dissociation (CID) experiments and density functional theory calculations at the B3LYP 6-31++G(d, p) level of theory was used to investigate the influence of the position of the radical site and the basicity of the amino acid residues in the radical cationic tripeptide analogs on their dissociation pathways. The CID spectra of two isomeric glycylglycyltryptophan radical cations-[GGW]?+ and [G?GW]+, with well-defined initial radical sites at the 3-methylindole ring and the N-terminal α-carbon atom, respectively-are significantly different. The former leads to the formation of [a3 ] H]?+, [c2 ] 2H]?+, and [z1 - H]?+ product ions through C-Cα and N-Cα peptide bond cleavages, while the latter leads to the predominant fragment ions of y1+, [b2 - H]?+, and [b3 - H]?+ via amide bond cleavages. After substitution of the central glycine residue of GGW with an arginine residue, however, the two isomers [G?RW]+ and [GRW]?+ produced almost identical CID spectra. The calculated energy barriers and microcanonical rate constants for isomerizations and competitive dissociations are in accordance with the perception that isomerizations between the GGW isomers could not compete with their fragmentations. For the radical cationic isomers, the presence of the highly basic arginine residue decreases the isomerization barriers (ca. 7-11 kcal/mol) and mediates facile hydrogen atom transfers-both along the peptide backbone and within the side chain residues-prior to subsequent dissociations. The effect of a basic amino acid residue on the isomerizations and dissociations of α-carbon-centered radical peptides also extends to distinctive Cβ-Cγ bond cleavages of isobaric leucine and isoleucine (Xle) residues. The CID spectra of [G?RXle]+ radical cations lead to the formation of characteristic product ions resulting from losses of ?CH(CH3)2 in [G?RL]+ and ?CH2CH3 in [G?RI]+ through Cβ-Cγ side-chain cleavages of (iso)leucine residues, allowing the two peptides to be distinguished. Finally, the first implementation of laser-induced dissociation (LID) on a hybrid quadrupole linear ion trap mass spectrometer is presented. After laser irradiation of mass-selected and -trapped ions in the quadrupole linear ion trap, LID spectra of [M]H]+ undergo both facile backbone and side-chain cleavages. These products are strikingly different from those formed in the CID spectra of [M]H]+, but are similar to those in the corresponding CID spectra of M?+. This approach provides an alternati

This dissertation, "Dissociation and Characterization of Cationic Radical Peptides" by Minjie, Xu, 许敏洁, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Gas phase fragmentations of cationic radical peptides provide important fundamental information that forms the basis for peptide sequencing by using mass spectrometry. Presenting results from low-energy collision-induced dissociation (CID) experiments and theoretical density functional theory (DFT) calculations in conjunction with Rice-Ramsperger-Kassel-Marcus modeling, this thesis describes some of the chemical properties, including the locations of the charge and radical sites that determine the gas-phase chemistry of peptide radical cations. The first Section (3.1) documents the dissociations of two isomeric glycylglycylarginine methyl ester radical cations, [G-GR-OMe]+ and [GG-R-OMe]+, with well-defined initial radical sites at the N-terminal and middle α-carbon atoms, respectively. These two isomers undergo similar fragmentations to form the y2+ ion and protonated allylguanidine; their identical CID spectra suggest that isomerization occurs prior to dissociation. DFT calculations at the B3LYP/6-31++G(d, p) level revealed that the proton is sequestered on the guanidine group of the side chain in the presence of a highly basic arginine residue, thereby decreasing the isomerization barriers among the α-carbon-centered radicals to approximately 36 kcal mol-1 (cf. 45 kcal mol-1 for the non-basic [GGG]-+ analogues) and facilitating the radical migration along the peptide backbone and subsequent dissociation reactions. The second section (3.2) describes an investigation into the specific effect of the N-terminal basic residue on selective Cα-C bond cleavage of aromatic-containing radical cationic peptides. Upon replacing the arginine residue of [R(G)n-2X(G)7-n]-+ by a less-basic lysine residue, forming [K(G)n-2X(G)7-n]-+ (X = Phe or Tyr; n = 2-7) analogues, the selective Cα-C peptide bond cleavage no longer occurs. The dissociations of the prototypical radical cationic tripeptides [RFG]-+ and [KFG]-+ at the second Cα-C peptide bonds of α-radical intermediates proceed with comparable barriers (ca. 33 and 35 kcal mol-1, respectively); the generation of the competitive [b2 - H]-+ fragment from [RFG]-+ (ca. 40 kcal mol-1) is much higher in energy than that from [KFG]-+ (ca. 27 kcal mol-1). Thus, the selective Cα-C bond cleavage product from [KFG]-+ can be overridden by the [b2 - H]-+ species in the absence of a basic N-terminal residue. Section (3.3) further examines the mechanistic roles of various α- andβ-carbon-centered radicals prior to Cα-C bond cleavage, leading to the observation of novel x-type radical fragments. DFT calculations and RRKM modeling of a prototypical π-radical cationic system, [AY]-+, suggested that direct Cα-C bond cleavage leading to the formation of the [x1 ] H]-+ species is thermodynamically comparable (ca. 16 kcal mol-1) with, but kinetically at least three-fold more favorable than, the well-characterized competitive formation of [c1 ] 2H]+ and [z1 - H]-+ species. This finding agrees well with the experimental yield of the [x1 ] H]-+ radical cation being higher than that of the minor [c1 ] 2H]+ species. DOI: 10.5353/th_b5185925 Subjects: Peptides - Analysis Cations

This dissertation, "Generation and Characterization of Cationic and Anionic Radical Peptides" by Ngor-wai, Lam, 林我威, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of the thesis entitled GENERATION AND CHARACTERIZATION OF CATIONIC AND ANIONIC RADICAL PEPTIDES Submitted by Lam Ngor Wai for the degree of Doctor of Philosophy at The University of Hong Kong in October 2006 Electron transfer is a fundamentally important chemical process. For example, oxidized radical products, derived from the oxidization or reduction of copper(II)-protein complex ions, have been proposed as key intermediates in a number of neurodegenerative disorders; but the mechanisms for their formation remain unclear. The gas phase dissociation of copper(II)-peptide complexes, II -2] [Cu (L)M] (L, ligand; M, peptide), has been demonstrated to generate peptide -+ radical cations (M ) through an electron transfer dissociation mechanism. Although such complexes are simple models for studying the fundamental parameters that govern the formation of peptide radical cations through single-electron transfer in the II -2+ absence of solvation, electron transfer from [Cu (L)] complexes is applicable only to a limited number of peptides, particularly those containing tyrosine, tryptophan, lysine, arginine, and histidine residues, when L is either a diethylenetriamine or 2,2 6,2-terpyridine ligand; for other peptides, competitive reaction channels predominate. This thesis describes the use of a prototypical system to explore the fundamental factors that govern the formation of peptide radical cations, and to achieve the efficient formation of aliphatic-only peptide radical cations. Copper(II) complexes were designed to allow exquisite control over the nature of the -+ competitive reactions. A wide variety of M radical cations were produced after altering the structure of the auxiliary ligand on the copper atom. In particular, the degree of steric hindrance about the auxiliary ligand is a major factor determining whether the electron transfer pathway predominates. After substituting the auxiliary ligand with more sterically encumbered 6,6-dibromo-2,2 6,2-terpyridine, 1,4,7-triazacyclononane or 1,4,7,10-tetraoxacyclododecane ligands, electron transfer reactions were facilitated tremendously to generate even the aliphatic-only tripeptide radical cations. The effect of the sterically constrained ligand is somewhat reminiscent of the entatic states of copper(II) ions in metalloproteins, in which constrained ligated copper(II) complexes facilitate electron transfer. The effects that non-zwitterionic peptide structures have on the formation of peptide radical cations were also examined. Formation of radical cations of aliphatic-only peptides was enhanced significantly when the peptide was forced to adopt a non-zwitterionic form. Collision-induced dissociation of chemically modified peptides was used to confirm the presence of both non-zwitterionic and zwitterionic -+ structures for the radical cations. The appearance of stable [a ] H] ions suggested that C -C bond cleavage of the amino acid side chains had been induced; this β γ feature allowed the unambiguous distinction of isomeric leucine and isoleucine residues in such peptides. An extension of the strategy allowed the production also of dicationic radical -2+ -- peptides [M ] H] and anionic radical peptides [M - 2H] from the fragmentations II -3+ III of [Cu (terpy)(M ] H)] (terpy = 2,2':6',2"-terpyridine) and [Mn (salen)(M - -- 2H)] [salen = N, N'-ethylenebis(salicylideneaminto)