An unambiguous and practical synthesis of Oroxylin A: a commonly misidentified flavone.

Oroxylin A, a major flavonoid in the extracts of Oroxylum indicum as well as Scutellaria baicalensis possesses useful medicinal properties. Many of the published routes claiming the synthesis of Oroxylin A (1) have in fact led to a regioisomer Negletein that was misinterpreted as Oroxylin A. In the present work, we describe a novel, straight-forward and scalable semi-synthetic approach for rapid access to the title compound, the structure of which is unambiguously secured by NMR and X-ray crystallographic analysis of a derivative. This work also encompasses the synthesis of a glycosylated derivative of Oroxylin A viz OAGME (2), which has marked pharmacological importance.

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.

Chemical Synthesis of Ubiquitin Chains.

Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di-ubiquitin chains.

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.

Dehydroalanine-based diubiquitin activity probes.

A strategy for the synthesis of dehydroalanine based diubiquitin activity probes is described. The site-specific introduction of dehydroalanine was achieved from diubiquitin bearing Cys residue near the scissile bond between two ubiquitins linked through Lys48, Lys63 or in a head to tail fashion. The probes were characterized for their activities with various deubiquitinases, which open new opportunities in studying deubiquitinases in various settings.

Modifying the vicinity of the isopeptide bond to reveal differential behavior of ubiquitin chains with interacting proteins: organic chemistry applied to synthetic proteins.

In every direction: Chemical protein synthesis allows the construction of 14 di-ubiquitin analogues modified in the vicinity of the isopeptide bond to examine their behavior with deubiquitinases and ubiquitin binding domains. The results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins.

Non-enzymatic synthesis of ubiquitin chains: where chemistry makes a difference.

Ubiquitination is a highly important posttranslational modification in eukaryotic cells where a target protein is conjugated to ubiquitin or a chain of ubiquitins via an isopeptide bond to trigger various cellular events such as proteasomal degradation. Rigorous investigations of the ubiquitin signal at the molecular level require homogeneous samples of ubiquitin chains in their free form or as anchored to a protein substrate in adequate quantities. The complexity of ubiquitin chains in terms of linkage types (owing to presence of seven Lys in ubiquitin) makes them difficult to prepare via enzymatic methods. Even more challenging is the attachment of these chains to a protein target at a selected site. This dearth is being filled by the recent developments of novel chemical tools that offer atomic level control over the synthesis for structural and functional studies. These emerging chemical approaches are discussed in this mini-review with focus on the preparation of ubiquitin chains to aid the ongoing efforts in understanding their role in the ubiquitin signal.

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.

Epimerization free synthesis of O-acyl isodipeptides employing COMU.

O-Acyl isodipeptides are prepared by coupling Boc-Ser/Thr-OBzl with Fmoc-Xaa-OH employing COMU, well known third generation peptide coupling agent. The reaction proceeds with high yield and the chemical homogeneity of the synthesized molecules were established via chiral HPLC analyses. The O-acyl isodipeptide units play crucial role in the success of ' click peptide' protocol employed for assembling ' difficult sequence' peptides.

Poly(vinyl)chloride supported palladium nanoparticles: catalyst for rapid hydrogenation reactions.

Palladium nanoparticles supported over poly(vinyl)chloride matrix (PVC-Pd(0)) are prepared through an efficient and inexpensive protocol. The catalyst has been characterized by XRD, SEM and TEM and its utility for the reduction of a range of functional groups as well as for the removal of some common protecting groups employed in peptide chemistry is demonstrated.

Isoselenocyanates derived from amino acid esters: an expedient synthesis and application to the assembly of selenoureidopeptidomimetics, unsymmetrical selenoureas and selenohydantoins.

An important class of organoselenium compounds-α-isoselenocyanato esters 4 has been prepared by a reaction of α-isocyano esters with elemental selenium powder. The reaction issimple, rapid and all the isoselenocyanates have been isolated as stable ones after chromatographic purification. These hitherto unreported classes of molecules would be useful building blocks for the preparation of variety of selenium containing peptidomimetics. In this study, the utility of the title molecules in the preparation of selenoureidopeptidomimetics 6, unsymmetrical selenoureas 8 and selenohydantoins 10 is demonstrated.