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

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

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag

The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and “off-targets” exhibiting a range of binding affinities, cellular abundances, and binding characteristics

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (<i>K</i>_D = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications

NanoBRETA Novel BRET Platform for the Analysis of Protein–Protein Interactions

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (<i>K</i>_D = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications