Chemical synthesis of phosphorylated ubiquitin and diubiquitin exposes positional sensitivities of e1-e2 enzymes and deubiquitinases.

Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63-linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1-E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal.

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.

Lewis acid mediated intramolecular C-C bond formation of alkyne-epoxide leading to six-membered nitrogen and oxygen heterocycles.

Intramolecular C-C bond formation of oxygen- and nitrogen-tethered alkynes and epoxide mediated by Lewis acid under ambient conditions is described. A simple procedure for the synthesis of 3,6- and 5,6-dihydropyrans and 3,4-dehydropiperidines from acyclic building blocks in good yields without using any transition metal is shown.

Rapid End-Group Modification of Polysaccharides for Biomaterial Applications in Regenerative Medicine.

Polysaccharides have emerged as important functional materials because of their unique properties such as biocompatibility, biodegradability, and availability of reactive sites for chemical modifications to optimize their properties. The overwhelming majority of the methods to modify polysaccharides employ random chemical modifications, which often improve certain properties while compromising others. On the other hand, the employed methods for selective modifications often require excess of coupling partners, long reaction times and are limited in their scope and wide applicability. To circumvent these drawbacks, aniline-catalyzed oxime formation is developed for selective modification of a variety of polysaccharides through their reducing end. Notably, it is found that for efficient oxime formation, different conditions are required depending on the composition of the specific polysaccharide. It is also shown how our strategy can be applied to improve the physical and functional properties of alginate hydrogels, which are widely used in tissue engineering and regenerative medicine applications. While the randomly and selectively modified alginate exhibits similar viscoelastic properties, the latter forms significantly more stable hydrogel and superior cell adhesive and functional properties. Our results show that the developed conjugation reaction is robust and should open new opportunities for preparing polysaccharide-based functional materials with unique properties.

Diastereoselective synthesis of substituted tetrahydrothiopyrans via (3,5)-thionium-ene cyclization reaction.

Tetrahydrothiopyrans have been efficiently synthesized in good yields with excellent diastereoselectivity from aldehydes and substituted 5-methylhex-4-ene-1-thiol via (3,5)-thionium-ene cyclization reaction mediated by boron trifluoride etherate.

An efficient synthesis of dihydro- and tetrahydropyrans via oxonium-ene cyclization reaction.

An efficient method has been developed for the synthesis of 2,3-dihydropyrans and 4-methylenetetrahydropyrans from aldehydes and substituted homoallyl alcohols in benzene mediated by boron trifluoride etherate in good yields. The reaction proceeds via oxonium-ene reaction.

Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags.

Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag via a phenylacetamidomethyl linker, which can be cleaved by PdCl2. The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.

Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins.

Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

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.

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.

Unveiling the mode of action of antibacterial labdane diterpenes from Alpinia nigra (Gaertn.) B. L. Burtt seeds.

The labdane diterpene, (E)-labda-8(17), 12-diene-15, 16-dial (compound A) and its epoxide analogue, (E)-8ß, 17-Epoxylabd-12-ene-15, 16-dial (compound B) were isolated from the seeds of Alpinia nigra for the first time. The antibacterial activities of both compounds were evaluated against three Gram-positive and four Gram-negative bacteria, and flow cytometric analysis revealed that these compounds caused significant damage to the bacterial cell membranes. Further, field emission scanning electron microscope imaging and cell leakage analysis confirmed that the labdane diterpenes were responsible for bacterial cell membrane damage and disintegration. Our findings provide new insight into the broad-spectrum effects of two natural labdane diterpenes that may be useful in the future development of herbal antibiotic products.