Estudos visando a síntese de compostos macrocíclicos e heterocíclicos via reações multicomponentes de Ugi

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2017.De modo geral, estruturas cíclicas ocupam um lugar de destaque na química orgânica e rotas sintéticas para a obtenção desta classe de moléculas são altamente desejáveis. É crescente o número de publicações em que reações multicomponente de Ugi são utilizadas visando à síntese de compostos macrocíclicos e heterocíclicos. Desta forma, neste trabalho foram propostas duas estratégias sintéticas distintas, ambas baseadas na reação de Ugi, para a construção de um acervo de compostos cíclicos. A primeira delas, baseada nos preceitos da MiBs (Multiple Multicomponent Macrocyclizations Including Bifunctional Building Blocks), em que componentes bifuncionais são necessários, visou a síntese de compostos macrocíclicos via reações consecutivas de Ugi empregando diácidos carboxílicos preparados a partir de reações de Ugi do tipo U-5C-4CR. A segunda estratégia buscou empregar um aldeído polifuncionalizado, produto de um rearranjo de Claisen em adutos de Morita-Baylis-Hillman, como um dos componentes em reações multicomponentes de Ugi. Esta abordagem permitiu a obtenção de compostos heterocíclicos estruturalmente distintos pela utilização de diferentes estratégias de ciclização.In general, cyclic structures occupy a prominent place in organic chemistry and synthetic routes for obtaining this class of molecules are highly desirable. There is a growing number of publications in which Ugi multi-component reactions are used for the synthesis of macrocyclic and heterocyclic compounds. In this work, two different synthetic strategies were proposed, both based on the Ugi reaction, for the construction of a collection of cyclic compounds. The first one, based on the precepts of the MiBs (Multiple Multicomponent Macrocyclics Including Bifunctional Building Blocks), in which bifunctional components are required, aimed the synthesis of macrocyclic compounds via consecutive Ugi reactions using carboxylic diacids prepared from U- 5C-4CR Ugi variant. The second strategy employ a polyfunctionalized aldehyde, product of a Claisen rearrangement in Morita-Baylis-Hillman adducts, as one of the components in Ugi multicomponent reactions. This approach allowed structurally distinct heterocyclic compounds readly obtained by the use of different cyclization strategies

Reagent for Evaluating Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) Performance in Bottom-Up Proteomic Experiments

We present a novel proteomic standard for assessing liquid chromatography–tandem mass spectrometry (LC-MS/MS) instrument performance, in terms of chromatographic reproducibility and dynamic range within a single LC-MS/MS injection. The peptide mixture standard consists of six peptides that were specifically synthesized to cover a wide range of hydrophobicities (grand average hydropathy (GRAVY) scores of −0.6 to 1.9). A combination of stable isotope labeled amino acids (¹³C and ¹⁵N) were inserted to create five isotopologues. By combining these isotopologues at different ratios, they span four orders of magnitude within each distinct peptide sequence. Each peptide, from lightest to heaviest, increases in abundance by a factor of 10. We evaluate several metrics on our quadrupole orbitrap instrument using the 6 × 5 LC-MS/MS reference mixture spiked into a complex lysate background as a function of dynamic range, including mass measurement accuracy (MMA) and the linear range of quantitation of MS1 and parallel reaction monitoring experiments. Detection and linearity of the instrument routinely spanned three orders of magnitude across the gradient (500 fmol to 0.5 fmol on column) and no systematic trend was observed for MMA of targeted peptides as a function of abundance by analysis of variance analysis (<i>p</i> = 0.17). Detection and linearity of the fifth isotopologue (i.e., 0.05 fmol on column) was dependent on the peptide and instrument scan type (MS1 vs PRM). We foresee that this standard will serve as a powerful method to conduct both intra-instrument performance monitoring/evaluation, technology development, and inter-instrument comparisons

Mass Spectrometry Compatible Surfactant for Optimized In-Gel Protein Digestion

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)­undecyloxy)­carbonylamino)­propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5–2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20–30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative <i>in vivo</i> cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative <i>in vivo</i> cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative <i>in vivo</i> cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative <i>in vivo</i> cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative <i>in vivo</i> cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization