23 research outputs found
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><sub>D</sub> = 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
NanoBRETA 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><sub>D</sub> = 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