8 research outputs found
Synthesis of N-Fmoc-ProtectedAmino Alkyl Thiocyanates/Selenocyanates and their Applicationin the Preparation of 5-Substituted S/Se-Linked Tetrazoles
A novel class of N-Fmoc-protected amino alkyl thiocy-
anates/selenocyanates has been prepared by thiocyanation/seleno-cyanation of the corresponding alkyl iodides. These thiocyanates/selenocyanates undergo a facile [2+3]-cycloaddition reaction with sodium azide to afford novel N-Fmoc amino alkylS/Se-linked tetra-zoles
Isoselenocyanates derived from Boc/Z-amino acids: synthesis, isolation,characterization, and application to the efficient synthesis of unsymmetrical selenoureas and selenoureidopeptidomimetics
Isoselenocyanates derived from Boc/Z-amino acids are prepared by the reaction of the corresponding isonitriles with selenium powder in presence of triethylamine at reflux. The utility of these new classes of isoselenocyanates in the preparation of selenoureidodipeptidomimetics possessing both amino as well as carboxy termini has been accomplished. The H-1 NMR analysis confirmed that the protocol involving the conversion of isonitriles to isoselenocyanates and their use as coupling agents in assembling selenour-eido
derivatives is free from racemization. (C) 2010 Elsevier Ltd. All rights reserved
Polymerization Behavior of a Bifunctional Ubiquitin Monomer as a Function of the Nucleophile Site and Folding Conditions
Biopolymers with repeating modules composed of either
folded peptides
or tertiary protein domains are considered some of the basic biomaterials
that nature has evolved to optimize for energy efficient synthesis
and unique functions. Such biomaterials continue to inspire scientists
to mimic their exceptional properties and the ways that nature adopts
to prepare them. Ubiquitin chains represent another example of nature’s
approach to use a protein-repeating module to prepare functionally
important biopolymers. In the current work, we utilize a novel synthetic
strategy to prepare bifunctional ubiquitin monomers having a C-terminal
thioester and a nucleophilic 1,2-aminothiol at a desired position
to examine their polymerization products under different conditions.
Our study reveals that such analogues, when subjected to polymerization
conditions under different folding states, afford distinct patterns
of polymerization products where both the dynamic and the tertiary
structures of the chains play important roles in such processes. Moreover,
we also show that the presence of a specific ubiquitin-binding domain,
which binds specifically to some of these chains, could interfere
selectively with the polymerization outcome. Our study represents
the first example of examining the polymerization of designed and
synthetic repeating modules based on tertiary protein domains and
affords early lessons in the design and synthesis of biomaterial.
In regards to the ubiquitin system, our study may have implications
on the ease of synthesis of ubiquitin chains with varying lengths
and types for structural and functional analyses. Importantly, such
an approach could also assist in understanding the enzymatic machinery
and the factors controlling the assembly of these chains with a desired
length
Nonenzymatic Polyubiquitination of Expressed Proteins
Ubiquitination
is one of the most ubiquitous posttranslational
modifications in eukaryotes and is involved in various cellular events
such as proteasomal degradation and DNA repair. The overwhelming majority
of studies aiming to understand ubiquitination and deubiquitination
have employed unanchored ubiquitin chains and mono-ubiquitinated proteins.
To shed light on these processes at the molecular level, it is crucial
to have facile access to ubiquitin chains linked to protein substrates.
Such conjugates are highly difficult to prepare homogenously and in
workable quantities using the enzymatic machinery. To address this
formidable challenge we developed new chemical approaches to covalently
attach ubiquitin chains to a protein substrate through its Cys residue.
A key aspect of this approach is the installation of acyl hydrazide
functionality at the C-terminus of the proximal Ub, which allows,
after ubiquitin chain assembly, the introduction of various reactive
electrophiles for protein conjugation. Employing α-globin as
a model substrate, we demonstrate the facile conjugation to K48-linked
ubiquitin chains, bearing up to four ubiquitins, through disulfide
and thioether linkages. These bioconjugates were examined for their
behavior with the USP2 enzyme, which was found to cleave the ubiquitin
chain in a similar manner to unanchored ones. Furthermore, proteasomal
degradation study showed that di-ubiquitinated α-globin is rapidly
degraded in contrast to the mono-ubiquitinated counterpart, highlighting
the importance of the chain lengths on proteasomal degradation. The
present work opens unprecedented opportunities in studying the ubiquitin
signal by enabling access to site-specifically polyubiquitinated proteins
with an increased size and complexity
Synthetic Uncleavable Ubiquitinated Proteins Dissect Proteasome Deubiquitination and Degradation, and Highlight Distinctive Fate of Tetraubiquitin
Various hypotheses have been proposed
regarding how chain length,
linkage type, position on substrate, and susceptibility to deubiquitinases
(DUBs) affect processing of different substrates by proteasome. Here
we report a new strategy for the chemical synthesis of ubiquitinated
proteins to generate a set of well-defined conjugates bearing an oxime
bond between the chain and the substrate. We confirmed that this isopeptide
replacement is resistant to DUBs and to shaving by proteasome. Analyzing
products generated by proteasomes ranked how chain length governed
degradation outcome. Our results support that (1) the cleavage of
the proximal isopeptide bond is not a prerequisite for proteasomal
degradation, (2) by overcoming trimming at the proteasome, tetraUb
is a fundamentally different signal than shorter chains, and (3) the
tetra-ubiquitin chain can be degraded with the substrate. Together
these results highlight the usefulness of chemistry to dissect the
contribution of proteasome-associated DUBs and the complexity of the
degradation process