Strategies for the Stereoselective Synthesis of Carbon Quaternary Centers via Transition Metal-Catalyzed Alkylation of Enolate Compounds

Notwithstanding advances in modern chemical methods, the selective installation of sterically encumbered carbon stereocenters, in particular all-carbon quaternary centers, remains an unsolved problem in organic chemistry. The prevalence of all-carbon quaternary centers in biologically active natural products and pharmaceutical compounds provides a strong impetus to address current limitations in the state of the art of their generation. This thesis presents four related projects, all of which share in the goal of constructing highly-congested carbon centers in a stereoselective manner, and in the use of transition-metal catalyzed alkylation as a means to address that goal.

The first research described is an extension of allylic alkylation methodology previously developed in the Stoltz group to small, strained rings. This research constitutes the first transition metal-catalyzed enantioselective α-alkylation of cyclobutanones. Under Pd-catalysis, this chemistry affords all–carbon α-quaternary cyclobutanones in good to excellent yields and enantioselectivities.

Next is described our development of a (trimethylsilyl)ethyl β-ketoester class of enolate precursors, and their application in palladium–catalyzed asymmetric allylic alkylation to yield a variety of α-quaternary ketones and lactams. Independent coupling partner synthesis engenders enhanced allyl substrate scope relative to allyl β-ketoester substrates; highly functionalized α-quaternary ketones generated by the union of our fluoride-triggered β-ketoesters and sensitive allylic alkylation coupling partners serve to demonstrate the utility of this method for complex fragment coupling.

Lastly, our development of an Ir-catalyzed asymmetric allylic alkylation of cyclic β-ketoesters to afford highly congested, vicinal stereocenters comprised of tertiary and all-carbon quaternary centers with outstanding regio-, diastereo-, and enantiocontrol is detailed. Implementation of a subsequent Pd-catalyzed alkylation affords dialkylated products with pinpoint stereochemical control of both chiral centers. The chemistry is then extended to include acyclic β-ketoesters and similar levels of selective and functional group tolerance are observed. Critical to the successful development of this method was the employment of iridium catalysis in concert with N-aryl-phosphoramidite ligands.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

New Catalytic Methods for the Preparation of Tryptophans and Pyrroloindolines: Total Synthesis of (+)-Naseseazines A and B and (–)-Aspergilazine A

Tryptophan and unnatural tryptophan derivatives are important building blocks for the total synthesis of natural products, as well as the development of new drugs, biological probes, and chiral small molecule catalysts. This thesis describes various catalytic methods for the preparation of tryptophan derivatives as well as their functionalization and use in natural product total synthesis.

Herein, the tandem Friedel–Crafts conjugate addition/asymmetric protonation reaction between 2-substituted indoles and methyl 2-acetamidoacrylate to provide enantioenriched trytophans is reported. This method inspired further work in the area of transition metal catalyzed arylation reactions. We report the development of the coppercatalyzed arylation of tryptamine and tryptophan derivatives. The utility of these transformations is highlighted in the five-step syntheses of the natural products (+)-naseseazine A and B. Further work on the development of a mild and general Larock indolization protocol to access unnatural tryptophans is also discussed.

Growth parameters for Lates niloticus (L.), Bagrus docmak (Forsskal), Oreochromis niloticus (L.), Clarias gariepinus (Burchell) and Synodontis species derived from tag returns

In a tagging experiment carried out in the Kenyan waters of Lake Victoria, an annual growth increment of 29 cm yr was obtained for Lates niloticus (L.). Growth parameters obtained using the von Bertalanffy model on the growth curve fitted by eye were L (inf.) = 122 cm yr and k = 0.26 yr. Data for other species tagged were inadequate to obtain meaningful results.

Isotope shift studies in the rare earths and lead

Isotope shifts of K_α1 x-ray transitions were measured for the Neodymium isotopes Nd 142, 143, 144, 145, 146, 148 and 150, the Samarium isotopes Sm 147, 148, 149, 150, 152 and 154, the Gadolinium isotopes Gd 154, 155, 156, 157, 158 and 160, the Dysprosium isotopes Dy 162 and 164, the Erbium isotopes Er 166, 168 and 170, the Hafnium isotopes Hf 178 and 180 and the Lead isotopes Pb 204, 206, 207 and 208. A curved crystal Cauchois spectrometer was used. The analysis of the measurement furnished the variation of the mean square charge radius of the nucleus, δ˂r²˃, for 23 isotope pairs. The experimental results were compared with theoretical values from nuclear models. Combining the x-ray shifts and the optical shifts in Nd and Sm yielded the optical mass shifts. An anomaly was observed in the odd-even shifts when the optical and the x-ray shifts were plotted against each other.

Prevenção da úlcera de pressão: resultados da ação educativa junto à equipe de enfermagem

Os pacientes hospitalizados, acamados ou restritos ao leito, apresentam uma gama de fatores de risco para o desenvolvimento das úlceras por pressão, diante disso, necessitam de uma assistência de enfermagem qualificada e eficaz na prevenção dessas lesões, a fim de evitar o desenvolvimento das mesmas. Desta forma, a pesquisa teve como objetivo investigar a influência da ação educativa junto à equipe de enfermagem das enfermarias de Clínica Médica na prevenção das úlceras por pressão. Tratou-se de um estudo quantitativo com delineamento descritivo com dispositivo de intervenção do tipo antes e depois, tendo como cenário as enfermarias de Clínica Médica de um hospital universitário na cidade do Rio de Janeiro. Os sujeitos foram os enfermeiros (n=15) e técnicos de enfermagem (n=18) da Clínica Médica e a unidade de medida do estudo foi o número de vezes que as ações de enfermagem, referentes à prevenção das úlceras por pressão e desempenhadas pelos sujeitos, foram observadas pela pesquisadora. Obtivemos 396 observações antes da ação educativa e 204 depois da ação educativa. A pesquisa apresentou três etapas: observação sistematizada antes da ação educativa, realização da ação educativa e observação sistematizada depois da ação educativa. Os resultados foram obtidos através da análise dos Check list antes e depois da ação educativa e constatamos, por meio de valores percentuais, melhoras significativas. A categoria sempre, considerada o valor ideal para todas as variáveis, apresentou aumento de 20 pontos percentuais entre as observações; a categoria frequentemente apresentou aumento de 07 pontos e a categoria raramente apresentou decréscimo de 27 pontos. Isso demonstra que a ação educativa atingiu o objetivo de sensibilizar a equipe de enfermagem quanto a prevenir as úlceras por pressão de forma qualificada. Concluímos que as úlceras por pressão continuam sendo um grande problema de saúde pública, merecendo atenção especial por parte da equipe de enfermagem. Acreditamos que a ação educativa é capaz de sensibilizar a equipe de enfermagem e motivar a transformação das ações e condutas, porém, ela é pontual e é apenas uma parte do processo educacional. Para alcançarmos a mudança comportamental e proporcionar uma assistência qualificada e eficiente aos pacientes em risco de desenvolver as úlceras por pressão, sugerimos o desenvolvimento de uma prática educativa contínua e permanente, capaz de manter a equipe de enfermagem em constante aperfeiçoamento.

Photobleaching process of xanthene dyes initiated by N-phenylglycine in the polyvinylalcohol film

The parameters such as quantum yield and molar absorption coefficients of the photoinitiator that are responsible for holographic sensitivity in photopolymer material are investigated with a single beam exposure experiment. The influence of exposure intensity, the concentrations of N-phenylglycine and dye on the photobleaching process of xanthenes dyes are presented. In addition, the effect of diphenyliodonium hexafluorophosphate salt on the quantum yield and molar absorption of xanthene dyes is studied. (c) 2005 Elsevier Ltd. All rights reserved.

双程掺镱光纤放大器的增益特征及优化

比较了掺钕光纤和掺镱光纤对1064nm光放大的特点．基于速率方程和传输方程，数值分析了在915nm泵浦下双程掺镱光纤放大器的增益特征，并且和传统的单程放大进行了比较．分析了信号光和泵浦光，以及粒子数沿着光纤方向的分布特点．最后对双程掺镱光纤放大器进行了优化．