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Rational design and directed evolution of probe ligases for site-specific protein labeling and live-cell imagingWhite, Katharine Alice January 2012 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Chemical fluorophores have superior photophysical properties to fluorescent proteins and are much smaller. However, in order to use these probes for live-cell protein imaging, highly specific labeling methods are required. Here, we will describe three efforts to re-engineer the E. coli enzyme, lipoic acid ligase (LplA), to catalyze the ligation of small-molecule probes onto recombinant proteins. We call this collection of methods the PRIME (PRobe Incorporation Mediated by Enzymes) methodologies. First, we describe the structure-guided mutagenesis of LplA and the identification of an LplA variant that can ligate a blue coumarin fluorophore onto a 13-amino acid LplA acceptor peptide (LAP2). This "coumarin ligase" can be used to image cellular proteins with high specificity, sensitivity, and minimal perturbation of the biology of the protein of interest. We also demonstrate how subpopulations of a protein of interest can be labeled using genetically targeted coumarin ligase. Second, we describe our attempts to use yeast display evolution and fluorescence activated cell sorting (FACS) to evolve a truncated LplA enzyme. The original truncated enzyme had severely decreased activity for LplA's natural substrate, lipoic acid. We created a 107 library of LplA mutants and, after four rounds of selection, produced a truncated LplA mutant with lipoylation activity equivalent to full-length LplA. We next sought to evolve activity for an unnatural small molecule probe, but found that this strategy was limited by both increased hydrophobic probe sticking when using the truncated enzyme and some enzyme-dependent nonspecificity. Finally, from a library of 107 LplA mutants, we evolved a full-length LplA capable of ligating an unnatural picolyl azide (pAz) substrate. We demonstrated improved activity of the "pAz ligase" in the secretory pathway and cell surface, two regions where coumarin ligase is inactive. This enzyme can also be used to image cell surface protein-protein interactions as well as label proteins as they are trafficked through the endoplasmic reticulum. These probe ligases will be useful tools for cell biologists interested in studying protein function or protein-protein interactions in the context of living cells. / by Katharine Alice White. / Ph.D.
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Synthesis and anticancer evaluation of agelastatin alkaloid derivatives and enantioselective total synthesis of aspidosperma alkaloidsAntropow, Alyssa Hope January 2018 (has links)
Thesis: Ph. D. in Organic Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references. / I. Synthesis and Evaluation of Agelastatin Derivatives as Potent Modulators for Cancer Invasion and Metastasis The synthesis of new agelastatin alkaloid derivatives and their anticancer evaluation in the context of the breast cancer microenvironment is described. A variety of Ni -alkyl and C5-ether agelastatin derivatives were accessed via application of our strategy for convergent imidazolone synthesis. We have discovered that agelastatin alkaloids are potent modulators for cancer invasion and metastasis at non-cytotoxic doses. We discuss the increased potency of (-)-agelastatin E as compared to (-)-agelastatin A in this capacity, in addition to identification of new agelastatin derivatives with activity that is statistically equivalent to (-)-agelastatin E. II. Enantioselective Synthesis of (-)-Vallesine: Late-stage C17-Oxidation via Complex Indole Boronation The first enantioselective total synthesis of (-)-vallesine via a strategy that features a late-stage regioselective C17-oxidation followed by a highly stereoselective transannular cyclization is described. The versatility of this approach is highlighted by divergent synthesis of the archetypal alkaloid of this family, (+)-aspidospermidine, and an A-ring oxygenated derivative (+)- deacetylaspidospermine, the precursor to (-)-vallesine, from a common intermediate. III. Enantioselective Total Synthesis of (-)-Jerantinine A from (-)-Melodinine P via Bio-Inspired A-Ring Oxidation The first enantioselective synthesis of (-)-melodinine P and its direct conversion to related alkaloid (-)-jerantinine A is described. A key para-aza-quinone methide pentacyclic intermediate enables A-ring to C-ring oxidation state transfer. Our synthesis is streamlined through the development of two multi-step single-pot procedures which proceed with high efficiency. We further demonstrate the utility ofpara-aza-quinone methide intermediates in our strategy for CI6-methoxylation which provides entry to the (-)-jerantinine alkaloid family. / by Alyssa Hope Antropow. / Ph. D. in Organic Chemistry
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Vapor-liquid equilibria from measurements of pressure temperature and over-all composition the system water-2butoxy ethanolWilson, Grant M January 1958 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1958. / Vita. / Includes bibliographical references (leaves 98-99). / by Grant McDonald Wilson. / Ph.D.
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The adsorption of molecular oxygen on Au/Ni(111) alloys and its oxidation of CO at 85 K / Molecular oxygen adsorbates on Au/Ni(111) alloy surfacesFischer, Jonathan David January 2010 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references. / Molecular oxygen is observed to adsorb on 0.2 - 0.6 ML Au/Ni(111) surface alloys. Molecular oxygen adsorbates are characterized by their 0-0 stretching frequencies of 740 cm', 851 cm' and 962 cm', as measured by high resolution electron energy loss spectroscopy (HREELS). The concentrations and frequencies of these species are determined as a function of Au coverage. The concentrations and frequencies of dissociatively chemisorbed molecular oxygen below 0.3 ML Au are measured using HREELS. A Monte Carlo simulation was developed to determine possible binding sites for the molecular and atomic oxygen adsorbates, but no reasonable geometric model was able to match the experimental data. The trends in concentration and frequency of the molecular oxygen adsorbates are caused by the downward shift of the Ni d band as Au is alloyed into the surface and the corresponding reduction in charge transfer from the metal surface to the antibonding orbitals of 02. Adsorbed molecular oxygen dissociates between 110 and 150 K into unreactive atomic oxygen. At Au coverage above 0.5 ML, the surface is observed to undergo a reconstruction at 300 K, leading to a new molecularly adsorbed oxygen species with a vibrational frequency of 1050 cm1. The total amount of oxygen adsorbed on the surface is quantified using Auger electron spectroscopy. An accurate algorithm for determining the Au coverage of Au/Ni( 111) surface alloys using Auger electron spectroscopy is described. Carbon monoxide is catalyically oxidized on the Au/Ni(1 11) alloy surface at 85 K. Molecularly adsorbed 02 species with vibrational frequencies of 851 cm' and 962 cm- 1 are identified as the reactants. At 115 K, CO reacts with the remaining molecular oxygen species with a vibrational frequency of 741 cm-'. The molecularly adsorbed oxygen stabilized on the reconstructed surface is also capable of reaction with CO at 85 K. The ability of Au/Ni(1 11) alloys to catalyze repeated oxidation reactions is demonstated, suggesting that Au/Ni(1 11) alloys may be a practical and effective catalyst for oxidizing CO to CO 2 at cryogenic to room temperature. / by Jonathan David Fischer. / Ph.D.
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The structural significance of doubly charged ions found in the mass spectra of some organic molecules.Vouros, Paul January 1965 (has links)
Massachusetts Institute of Technology. Dept. of Chemistry. Thesis. 1965. Ph.D. / Ph.D.
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Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductaseTong, Wing Hang January 1996 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996. / Includes bibliographical references. / by Wing Hang Tong. / Ph.D.
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Photochemical reductive elimination of halogen from transition metal complexesCook, Timothy R. (Timothy Raymond), 1982- January 2010 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references. / This thesis is focused on the synthesis and study of transition metal complexes that undergo halogen elimination when irradiated with UV and visible light. This chemistry is relevant for solar energy storage schemes in which simple substrates such as HX (X = Cl, Br, OH) can be split to H2 and X2 in thermodynamically uphill photocycles. The activation of strong M-X bonds is a key requirement for efficient photocatalysts and represents the most challenging step of HX splitting schemes. Studies have focused on the design and characterization of late-metal phosphine complexes with an emphasis on chemical oxidation with halogen to give stable, halide-rich metal centers, and the subsequent photochemistry of the resulting complexes. The primary goal of this work was to increase the photo-efficiency of M-X bond activation as (1) the low quantum yield of halogen elimination had been implicated as the governing step determining the efficiency of previously reported HX splitting catalysts and (2) mechanistic studies on the nature of halogen elimination using laser kinetic techniques require a substantial concentration of transient photointermediates for signal to be observed. These targets spurred the development of a number of late-metal homo- and heterobimetallic complexes which undergo highly efficient halogen elimination, as well as the first reports of authentic X2 reductive elimination from a transition metal center and direct observation of M-X bond activation using transient absorption spectroscopy. / by Timothy R. Cook. / Ph.D.
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Nickel-catalyzed reductive coupling reactions of 1,6-enynes and the total synthesis of (+)-acutiphycinMoslin, Ryan Thomas McLeod January 2007 (has links)
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007. / Vita. / Includes bibliographical references. / Nickel-Catalyzed Reductive Coupling Reactions of Aldehydes and Chiral 1,6-Enynes. A study of nickel-catalyzed reductive coupling reactions of aldehydes and chiral 1,6-enynes has provided evidence for stereospecific ligand substitution from a planar three-coordinate nickel species as a plausible explanation of regioselectivity in the nickel-catalyzed reductive coupling of aldehydes and 1,6-enynes. In the absence of a phosphine additive, high regioselectivity and high diastereoselectivity are obtained as a direct result of coordination of both the alkyne and the olefin to the metal center during the C-C bond-forming step. ... Total Synthesis of (+)-Acutiphycin. Synthetic studies toward the total synthesis of (+)-acutiphycin led to, and were in turn further developed by, the study of nickel-catalyzed reductive coupling reactions of 1,6-enynes and aldehydes. ... Ultimately, though not employing the nickel-catalyzed reaction, a highly convergent total synthesis of (+)-acutiphycin featuring an intermolecular SmI2-mediated Reformatsky coupling reaction and macrolactonization initiated by a retro-ene reaction of an alkoxyalkyne was developed. The resulting synthesis was 18 steps in the longest linear sequence from either methyl acetoacetate or isobutyraldehyde. ... / by Ryan Thomas McLeod Moslin. / Ph.D.
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Investigation of freezing properties of polar statospheric cloud particles with optical microscopyNg, Huey Pin January 1997 (has links)
Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997. / Includes bibliographical references (leaves 96-100). / by Huey Pin Ng. / M.S.
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Elucidation of the pathways responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine in bacterial pathogensMorrison, Michael James January 2014 (has links)
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2014. / Cataloged from PDF version of thesis. "February 2014." / Includes bibliographical references. / The highly-modified, bacterial sugar N,N'-diacetylbacillosamine (diNAcBac) has been implicated in the pathogenicity of certain microbes through its incorporation onto various protein virulence factors. In particular, diNAcBac is found at the reducing end of glycans in both asparagine (N-linked) and serine/threonine (0-linked) protein glycosylation pathways. The second and third chapters examine the O-linked protein glycosylation pathway responsible for the biosynthesis of the UDP-diNAcBac nucleotide sugar in Neisseria gonorrhoeae and Acinetobacter baumannii. UDP-diNAcBac is biosynthesized from UDP-N-acetylglucosamine through the action of a dehydratase, aminotransferase, and acetyltransferase. Specifically, these enzymes are purified, biochemically characterized, and compared to the N-linked pathway proteins from Campylobacterjejuni. Furthermore, the substrate specificity of the A. baumannii phosphoglycosyltransferase that catalyzes the transfer of UDP-diNAcBac onto undecaprenylphosphate is determined. The fourth chapter explores the structural characterization of the acetyltransferases from the O-linked protein glycosylation pathways in N. gonorrhoeae (PglB-ATD) and A. baumannii (Weel). These enzymes are members of the left-handed [beta]-helix family and are responsible for the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-[alpha]-D-glucose (UDP-4-amino) to produce UDP-diNAcBac. Based upon these structures, a series of active site mutations are generated and kinetically characterized for both the AcCoA and UDP-4-amino substrates. These results suggest that although each enzyme catalyzes the acetyltransferase reaction with identical substrates, key residues within the binding pockets can lead to a diverse set of catalytic efficiencies. The final three chapters investigate the inhibition of UDP-diNAcBac pathway enzymes in C. jejuni, N. gonorrhoeae, and A. baumannii. The fifth chapter explores a fragment-based approach to identify small molecules that inhibit the aminotransferase in C. jejuni. To this end, a crystal structure of this protein is solved in complex with a fragment molecule and analogs of this compound synthesized. The sixth chapter identifies small molecule acetyltransferase inhibitors through a high-throughput screening effort in collaboration with the Broad Institute. Lastly, the seventh chapter describes a fragment-based approach to establish small molecule inhibitors for the acetyltransferase from N gonorrhoeae. / by Michael James Morrison. / Ph. D.
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