CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.

Multiple sulfur isotope fractionations in inorganic aqueous systems

New constraints on isotope fractionation factors in inorganic aqueous sulfur systems based on theoretical and experimental techniques relevant to studies of the sulfur cycle in modern environments and the geologic rock record are presented in this dissertation. These include theoretical estimations of equilibrium isotope fractionation factors utilizing quantum mechanical software and a water cluster model approach for aqueous sulfur compounds that span the entire range of oxidation state for sulfur. These theoretical calculations generally reproduce the available experimental determinations from the literature and provide new constraints where no others are available. These theoretical calculations illustrate in detail the relationship between sulfur bonding environment and the mass dependence associated with equilibrium isotope exchange reactions involving all four isotopes of sulfur. I additionally highlight the effect of isomers of protonated compounds (compounds with the same chemical formula but different structure, where protons are bound to either sulfur or oxygen atoms) on isotope partitioning in the sulfite (S4+) and sulfoxylate (S2+) systems, both of which are key intermediates in oxidation-reduction processes in the sulfur cycle. I demonstrate that isomers containing the highest degree of coordination around sulfur (where protonation occurs on the sulfur atom) have a strong influence on isotopic fractionation factors, and argue that isomerization phenomenon should be considered in models of the sulfur cycle. Additionally, experimental results of the reaction rates and isotope fractionations associated with the chemical oxidation of aqueous sulfide are presented. Sulfide oxidation is a major process in the global sulfur cycle due largely to the sulfide-producing activity of anaerobic microorganisms in organic-rich marine sediments. These experiments reveal relationships between isotope fractionations and reaction rate as a function of both temperature and trace metal (ferrous iron) catalysis that I interpret in the context of the complex mechanism of sulfide oxidation. I also demonstrate that sulfide oxidation is a process associated with a mass dependence that can be described as not conforming to the mass dependence typically associated with equilibrium isotope exchange. This observation has implications for the inclusion of oxidative processes in environmental- and global-scale models of the sulfur cycle based on the mass balance of all four isotopes of sulfur. The contents of this dissertation provide key reference information on isotopic fractionation factors in aqueous sulfur systems that will have far-reaching applicability to studies of the sulfur cycle in a wide variety of natural settings.

Diversity in Catalytic Reactions of Propargylic Diazoesters

Abstract Title of Document: Diversity in Catalytic Reactions of Propargylic Diazoesters Huang Qiu, Doctor of Philosophy, 2016 Directed By: Professor Michael P. Doyle, Department of Chemistry and Biochemistry Propargylic aryldiazoesters, which possess multiple reactive functional groups in a single molecule, were expected to undergo divergent reaction pathways as a function of catalysts. A variety of transition metal complexes including rhodium(II), palladium(II), silver(I), mercury(II), copper(I and II), and cationic gold (I) complexes have been examined to be effective in the catalytic domino reactions of propargylic aryldiazoesters. An unexpected Lewis acid catalyzed pathway was also discovered by using FeCl3 as the catalyst. Under the catalysis of selected gold catalysts, propargylic aryldiazoesters exist in equilibrium with 1-aryl-1,2-dien-1-yl diazoacetate allenes that are rapidly formed at room temperature through 1,3-acyloxy migration. The newly formed allenes further undergo a metal-free rearrangement in which the terminal nitrogen of the diazo functional group adds to the central carbon of the allene initiating a sequence of bond forming reactions resulting in the production of 1,5-dihydro-4H-pyrazol-4-ones in good yields. These 1,5-dihydro-4H-pyrazol-4-ones undergo intramolecular 1,3-acyl migration to form an equilibrium mixture or quantitatively transfer the acyl group to an external nucleophile with formation of 4-hydroxypyrazoles. In the presence of a pyridine-N-oxide, both E- and Z-1,3-dienyl aryldiazoacetates are formed in high combined yields by Au(I)-catalyzed rearrangement of propargyl arylyldiazoacetates at short reaction times. Under thermal reactions the E-isomers form the products from intramolecular [4+2]-cycloaddition with H‡298 = 15.6 kcal/mol and S‡298= -27.3 cal/ (mol•degree). The Z-isomer is inert to [4+2]-cycloaddition under these conditions. The Hammett relationships from aryl-substituted diazo esters ( = +0.89) and aryl-substituted dienes ( = -1.65) are consistent with the dipolar nature of this transformation. An unexpected reaction for the synthesis of seven-membered conjugated 1,4-diketones from propargylic diazoesters with unsaturated imines was disclosed. To undergo this process vinyl gold carbene intermediates generated by 1,2-acyloxy migration of propargylic aryldiazoesters undergo a formal [4+3]-cycloaddition, and the resulting aryldiazoesters tethered dihydroazepines undergo an intricate metal-free process to form observed seven-membered conjugated 1,4-diketones with moderate to high yields.