Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010. / Committee Chair: Bridgette Barry; Committee Member: David Collard; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Jake Soper; Committee Member: Mira Josowicz. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 270-290). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Understanding the dissociation of odd-electron peptide radical cations is of great importance for the analytical applications of biological mass spectrometry because their diverse array of fragmentation pathways provides structural information to supplement that from even-electron protonated peptides—allowing peptide sequencing and, ultimately, protein identification. Nevertheless, the mechanisms of peptide radical formation and dissociation remain largely unexplored. In the studies reported in this Thesis, peptide radical cations (M?+) were generated through one-electron transfer (ET) in collision-induced dissociation (CID) of [CuII(L)M]?2+ (L = auxiliary ligand; M = peptide) complexes. Competitive dissociative pathways were circumvented experimentally through judicious selection of the macrocyclic auxiliary ligand, allowing the formation of a broad range of M?+ species. Chapter 3.1 examines the competition between proton transfer (PT) and ET within [CuII(L)His]?2+ complexes with L = dien (an open-chain ligand), or L = 9-aneN3 (the macrocyclic analogue of dien). Density functional theory (DFT) calculations revealed that macrocyclic ligand (9-aneN3) facilitates M?+ formation by maintaining similar ET barriers with open-chain ligand (dien), but substantially increasing PT barriers. Studying and understanding the fragmentations of M?+ species is fundamentally important and a formidable challenge—both charge-directed and radical-driven fragmentations play important roles, in a competitive manner, in the dissociations of M?+ species. Chapters 3.2-3.4 were built upon successful gas phase syntheses of a wide variety of M?+ species. Chapter 3.2 reports the novel Cβ–Cγ bond cleavage of tryptophan residues in the dissociations of various tryptophan-containing M?+ species, resulting in a neutral 116-Da loss; this process is an α-radical–induced fragmentation. Substitution of the tryptophan residue by a 1-methyltryptophan residue revealed that the 116-Da neutral species is a radical with an unpaired electron on the indole nitrogen atom. Chapter 3.3 describes a systematic examination of tryptophan-containing model systems, both with and without basic residues, to unveil the mechanisms of Cβ–Cγ bond cleavages. M?+ species containing non-basic residues undergo protonation of the γ-carbon atom of the tryptophan residue, thereby weakening the Cβ–Cγ bond and facilitating its cleavage. The formation of [1H-indole]?+ (m/z 117) or [M – CO2 – 116]+ ions is a competition between two incipient fragments for the proton in a dissociating proton-bound dimer. In basic residue containing M?+ species, the proton is tightly sequestered by the basic side chain, resulting in more accessible radical migration barriers prior to subsequent bond cleavages; DFT calculations supported the notion that the charge-remote radical-driven pathway is more favorable than the proton-driven process by 6.2 kcal/mol. Selective radical-induced fragmentations were then used to investigate the radical propagation processes occurring via hydrogen atom transfers—in particular, for Cα–C bond cleavages leading to the formation of an+ ions. The energetics and kinetics of the dissociations of [RGn–2FG7–n – CO2]?+ (n = 2–6) with well-defined C-terminal α-radicals were determined by RRKM modeling of surface-induced dissociation experiments and DFT calculations, revealing that radical propagations in peptide radical cations are not necessarily stepwise processes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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 alternative means of identifying peptide sequence in shogun proteomic analysis. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

abstract / Chemistry / Doctoral / Doctor of Philosophy

Class B GPCRs are emerging target in drug research. Currently these receptors serve as drug targets for several drug discovery companies and more than 50 percent of the drugs in the market targets GPCRs. Secretin receptor is found to be expressed in various tissues. Secretin regulates many bodily functions from energy to water homeostasis through both central and peripheral system. Though it holds a history of 100 years, the major drawback is its structural insights. In evidence of its integrated role in physiology as a potential target, the lookout for a novel agonist and / or antagonist for secretin receptor is initiated. As this target is in the primary state of drug research, it is also necessary to develop the appropriate screening platforms. Due to the lack of experimental structure of secretin receptor-ligand, a 3D virtual homology model is developed using multiple template approach. Besides virtual docking, a non-radioactive FRET competitive binding assay is also developed and substantiated to enable the receptor-ligand interaction studies. Both peptide and non-peptide analogs were screened for virtual docking, in vitro binding and functional response. For the peptide analogs, the modifications were made either in the N or C terminal portion of the peptide based on the previous findings that C-terminal portion is involved in receptor binding followed by allosteric modifications and N-terminal portion is involved in activation. These peptide analogs exhibited binding affinity in the virtual model. Paradoxically it did not exhibit in vitro binding as predicted. Along with this, the agonistic and antagonistic functional responses of these peptide analogs were also found to be negative. SPECS natural product library of 500 non-peptide analogs were screened virtually against secretin receptor and 32 hits were identified. Of these hits glycyrrhizin’s functions were comparable to secretin was screened for receptor binding and functional response. These in vitro assays did not exert anything positive; however an IP-GTT on WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice with acute treatment of glycyrrhizin at 10 mg/kg and chronic treatment of 5 mg/kg exhibited an interesting profile with negligible effect on 〖SCT〗^(-/- )mice whereas in WT and 〖SCTR〗^(-/-) mice it displayed a better profile with improved glucose tolerance. The chronic study serum analysis on day 28 exhibited substantial reduction in blood glucose while significant increase in serum secretin and insulin levels. As glycyrrhizin promotes secretin secretion, its acute effect on blood pressure in WT mice was also analyzed at 10 mg/kg; remarkably exhibited a significant drop in blood pressure. In summary modifications in the peptide analogs lead to instability in the receptor-ligand binding complex in the in vitro system leading to loss of binding efficiency. In case of non-peptides, though glycyrrhizin could not exhibit in vitro response, its supplementary mechanism through secretin pathway of increased secretin release is confirmed using the WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice. The hypotensive effect with an acute treatment in WT is also revealed. Discovery of this new mechanism of an old drug could broaden the research for a new class of drug, “secretin sensitizers / promoters”. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Not available / text

Not available