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

Improving Proteomics Mass Accuracy by Dynamic Offline Lock Mass

Several methods to obtain low-ppm mass accuracy have been described. In particular, online or offline lock mass approaches can use background ions, produced by electrospray under ambient conditions, as calibrants. However, background ions such as protonated and ammoniated polydimethylcyclosiloxane ions have relatively weak and fluctuating intensity. To address this issue, we implemented dynamic offline lock mass (DOLM). Within every MS1 survey spectrum, DOLM dynamically selected the strongest <i>n</i> background ions for statistical treatments and <i>m</i>/<i>z</i> recalibration. We systematically optimized the mass profile abstraction method to find one single <i>m</i>/<i>z</i> value to represent an ion and the number of calibrants. To assess the influence of the intensity of the analyte ions, we used tandem mass spectroscopy (MS/MS) datasets obtained from MudPIT analyses of two protein samples with different dynamic ranges. DOLM outperformed both external mass calibration and offline lock mass that used predetermined calibrant ions, especially in the low-ppm range. The unique dynamic feature of DOLM was able to adapt to wide variations in calibrant intensities, leading to averaged mass error center at 0.03 ± 0.50 ppm for precursor ions. Such consistently tight mass accuracies meant that a precursor mass tolerance as low as 1.5 ppm could be used to search or filter post-search DOLM-recalibrated MS/MS datasets

Improving Proteomics Mass Accuracy by Dynamic Offline Lock Mass

Several methods to obtain low-ppm mass accuracy have been described. In particular, online or offline lock mass approaches can use background ions, produced by electrospray under ambient conditions, as calibrants. However, background ions such as protonated and ammoniated polydimethylcyclosiloxane ions have relatively weak and fluctuating intensity. To address this issue, we implemented dynamic offline lock mass (DOLM). Within every MS1 survey spectrum, DOLM dynamically selected the strongest <i>n</i> background ions for statistical treatments and <i>m</i>/<i>z</i> recalibration. We systematically optimized the mass profile abstraction method to find one single <i>m</i>/<i>z</i> value to represent an ion and the number of calibrants. To assess the influence of the intensity of the analyte ions, we used tandem mass spectroscopy (MS/MS) datasets obtained from MudPIT analyses of two protein samples with different dynamic ranges. DOLM outperformed both external mass calibration and offline lock mass that used predetermined calibrant ions, especially in the low-ppm range. The unique dynamic feature of DOLM was able to adapt to wide variations in calibrant intensities, leading to averaged mass error center at 0.03 ± 0.50 ppm for precursor ions. Such consistently tight mass accuracies meant that a precursor mass tolerance as low as 1.5 ppm could be used to search or filter post-search DOLM-recalibrated MS/MS datasets

Improving Proteomics Mass Accuracy by Dynamic Offline Lock Mass

Several methods to obtain low-ppm mass accuracy have been described. In particular, online or offline lock mass approaches can use background ions, produced by electrospray under ambient conditions, as calibrants. However, background ions such as protonated and ammoniated polydimethylcyclosiloxane ions have relatively weak and fluctuating intensity. To address this issue, we implemented dynamic offline lock mass (DOLM). Within every MS1 survey spectrum, DOLM dynamically selected the strongest <i>n</i> background ions for statistical treatments and <i>m</i>/<i>z</i> recalibration. We systematically optimized the mass profile abstraction method to find one single <i>m</i>/<i>z</i> value to represent an ion and the number of calibrants. To assess the influence of the intensity of the analyte ions, we used tandem mass spectroscopy (MS/MS) datasets obtained from MudPIT analyses of two protein samples with different dynamic ranges. DOLM outperformed both external mass calibration and offline lock mass that used predetermined calibrant ions, especially in the low-ppm range. The unique dynamic feature of DOLM was able to adapt to wide variations in calibrant intensities, leading to averaged mass error center at 0.03 ± 0.50 ppm for precursor ions. Such consistently tight mass accuracies meant that a precursor mass tolerance as low as 1.5 ppm could be used to search or filter post-search DOLM-recalibrated MS/MS datasets

Improving Proteomics Mass Accuracy by Dynamic Offline Lock Mass

Several methods to obtain low-ppm mass accuracy have been described. In particular, online or offline lock mass approaches can use background ions, produced by electrospray under ambient conditions, as calibrants. However, background ions such as protonated and ammoniated polydimethylcyclosiloxane ions have relatively weak and fluctuating intensity. To address this issue, we implemented dynamic offline lock mass (DOLM). Within every MS1 survey spectrum, DOLM dynamically selected the strongest <i>n</i> background ions for statistical treatments and <i>m</i>/<i>z</i> recalibration. We systematically optimized the mass profile abstraction method to find one single <i>m</i>/<i>z</i> value to represent an ion and the number of calibrants. To assess the influence of the intensity of the analyte ions, we used tandem mass spectroscopy (MS/MS) datasets obtained from MudPIT analyses of two protein samples with different dynamic ranges. DOLM outperformed both external mass calibration and offline lock mass that used predetermined calibrant ions, especially in the low-ppm range. The unique dynamic feature of DOLM was able to adapt to wide variations in calibrant intensities, leading to averaged mass error center at 0.03 ± 0.50 ppm for precursor ions. Such consistently tight mass accuracies meant that a precursor mass tolerance as low as 1.5 ppm could be used to search or filter post-search DOLM-recalibrated MS/MS datasets

Improving Label-Free Quantitative Proteomics Strategies by Distributing Shared Peptides and Stabilizing Variance

In a previous study, we demonstrated that spectral counts-based label-free proteomic quantitation could be improved by distributing peptides shared between multiple proteins. Here, we compare four quantitative proteomic approaches, namely, the normalized spectral abundance factor (NSAF), the normalized area abundance factor (NAAF), normalized parent ion intensity abundance factor (NIAF), and the normalized fragment ion intensity abundance factor (NFAF). We demonstrate that label-free proteomic quantitation methods based on chromatographic peak area (NAAF), parent ion intensity in MS1 (NIAF), and fragment ion intensity (NFAF) are also improved when shared peptides are distributed on the basis of peptides unique to each isoform. To stabilize the variance inherent to label-free proteomic quantitation data sets, we use cyclic-locally weighted scatter plot smoothing (LOWESS) and linear regression normalization (LRN). Again, all four methods are improved when cyclic-LOWESS and LRN are applied to reduce variation. Finally, we demonstrate that absolute quantitative values may be derived from label-free parameters such as spectral counts, chromatographic peak area, and ion intensity when using spiked-in proteins of known amounts to generate standard curves

Engineering of iSOX2-DAOY medulloblastoma cells to identify SOX2-associated proteins.

<p>(A) Schematic diagram of the lentiviral system used to introduce a Dox-inducible, epitope tagged SOX2 into DAOY MB cells. Two lentiviral vectors were used to introduce constitutively expressed reverse-tet transactivator (rtTA) and inducible (fs)SOX2 [labeled: Flag-SOX2]. (B) Protocol used to isolate SOX2-protein complexes from DAOY MB cells for downstream MudPIT analysis. (C) Western blot analysis probing for SOX2 with a Sox2 antibody. The level of (fs)SOX2 was compared to the level of endogenous SOX2, which was set to 1. (D) Silver stain demonstrating enrichment of proteins following the induction of (fs)SOX2 with Dox and immunoprecipitation with M2-beads. The prominent band observed at ∼45 kDa is a common contaminant when M2-beads are used for immunoprecipitation. *The estimated position of (fs)SOX2 is indicated by an arrowhead. (E) Western blot analysis probing for Flag-SOX2 to verify immunoprecipitation using M2-beads.</p

The SOX2-Interactome in Brain Cancer Cells Identifies the Requirement of MSI2 and USP9X for the Growth of Brain Tumor Cells

<div><p>Medulloblastomas and glioblastomas, the most common primary brain tumors in children and adults, respectively, are extremely difficult to treat. Efforts to identify novel proteins essential for the growth of these tumors may help to further our understanding of the biology of these tumors, as well as, identify targets for future therapies. The recent identification of multiple transcription factor-centric protein interaction landscapes in embryonic stem cells has identified numerous understudied proteins that are essential for the self-renewal of these stem cells. To identify novel proteins essential for the fate of brain tumor cells, we examined the protein interaction network of the transcription factor, SOX2, in medulloblastoma cells. For this purpose, Multidimensional Protein Identification Technology (MudPIT) identified >280 SOX2-associated proteins in the medulloblastoma cell line DAOY. To begin to understand the roles of SOX2-associated proteins in brain cancer, we focused on two SOX2-associated proteins, Musashi 2 (MSI2) and Ubiquitin Specific Protease 9x (USP9X). Recent studies have implicated MSI2, a putative RNA binding protein, and USP9X, a deubiquitinating enzyme, in several cancers, but not brain tumors. We demonstrate that knockdown of MSI2 significantly reduces the growth of DAOY cells as well as U87 and U118 glioblastoma cells. We also demonstrate that the knockdown of USP9X in DAOY, U87 and U118 brain tumor cells strongly reduces their growth. Together, our studies identify a large set of SOX2-associated proteins in DAOY medulloblastoma cells and identify two proteins, MSI2 and USP9X, that warrant further investigation to determine whether they are potential therapeutic targets for brain cancer.</p></div

Knockdown of MSI2 in U87 GB cells.

<p>(A) Western blot analysis of MSI2 levels in U87 nuclear extracts 96 hours after infection with Scrambled or MSI2 shRNA lentiviruses. MSI2 levels are quantified, with levels found in the Scrambled control set to 1.00. (B) Cell growth was examined in triplicate by MTT assay 5 days after being plated at 1.5×10⁴ cells per well of a 12-well plate. The data shown are averages relative to the Scramble control. Error bars represent standard deviation. P values were determined by student t-test and found to be <.01 for both MSI2 shRNA 1 and 2. (C) Photomicrographs of U87 GB cells following infection with either non-specific (Scrambled) or MSI2 targeting (No. 1, No. 2) shRNA lentiviruses. Cells were infected on Day 0, selected using medium supplemented with puromycin on Day 1 and refed fresh medium on Day 3. Cells were photographed on day 6 after infection.</p

Knockdown of USP9X in U87 GB cells.

<p>(A) Western blot analysis to verify the knockdown of USP9X in U87 GB cells following infection with lentiviruses to introduce constitutively active shRNA against USP9X transcripts. Nuclear and cytoplasmic protein fractions were prepared 4 days after infecting cells with lentiviruses. USP9X levels are quantified, and levels in the Scrambled control are set to 1.00. (B) Cell growth was examined in triplicate by MTT assay 5 days after being plated at 1.5×10⁴ cells per well of a 12-well plate. The data shown are averages relative to the Scramble control. Error bars represent standard deviation. P values were determined by student t-test and found to be <.01 for both USP9X shRNA 1, 2 and 3. (C) Photomicrographs of U87 GB cells following knockdown of USP9X using lentiviral delivered shRNA constructs against USP9X transcripts. On day 0, cells were infected with USP9X shRNA lentiviruses. Beginning on day 1, infected cells were selected using puromycin for 48 hours. On day 4, cells were passaged into fresh culture flasks (5×10⁵ cells per flask) and photographed on day 8 post-infection.</p