RESUMEN
Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to obtain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-terminal Cys in the middle fragments, which bear simultaneously the two reactive groups, i.e., N-terminal Cys and C-terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protecting group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example, the thiazolidine removal step is very slow (>8 h), and in some cases the applied conditions lead to undesired side reactions. Here we show that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palladium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficiency. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach expands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of synthetically challenging proteins from multiple fragments.
Asunto(s)
Cisteína/química , Paladio/química , Proteínas/síntesis química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Tiazolidinas/químicaRESUMEN
The chemical synthesis of a protein from four fragments or more applying native chemical ligation could be achieved stepwise, in one-pot, convergently, or on a solid support. With the increasing demands of applying protein synthesis to highly complex targets, examining these approaches becomes essential to achieve highly efficient synthesis. Different chemical synthetic strategies are compared for the preparation of the H2B protein having different post-translational modifications. The analogues include H2B that is ubiquitinated at Lys34, Lys120, glycosylated at Ser112, and doubly modified with ubiquitin and N-acetylglucosamine. This study demonstrates that the applied convergent strategy for the synthesis of most of these complex targets was better than the one-pot approach in terms of yield and purity. Some guidelines are offered for future synthetic endeavors of similar challenging proteins.
Asunto(s)
Acetilglucosamina/química , Histonas/síntesis química , Ubiquitina/química , Ubiquitinación , Glicosilación , Histonas/química , Conformación MolecularRESUMEN
Presented here is a solid phase chemical ligation strategy employing native chemical ligation and the commercially available Rink-amide linker as a key element in our approach. The method was applied for the synthesis of histone H2B, which sets the ground for the rapid preparation of posttranslationally modified analogues of this protein.
Asunto(s)
Amidas/química , Histonas/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Secuencia de Aminoácidos , Histonas/química , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Alanina/análogos & derivados , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitinas/metabolismo , Alanina/química , Secuencia de Aminoácidos , Humanos , Lisina/química , Modelos Moleculares , Estructura Molecular , Conformación ProteicaRESUMEN
Hybrid peptidic oligomers comprising natural and unnatural amino acid residues that can exhibit biomolecular folding and hydrogen-bonding mimicry have attracted considerable interest in recent years. While a variety of hybrid peptidic helices have been reported in the literature, other secondary structural patterns such as γ-turns and ribbons have not been well explored so far. The present work reports the design of novel periodic γ-turns in the oligomers of 1:1 natural-α/unnatural trans-ß-norborenene (TNAA) amino acid residues. Through DFT, NMR, and MD studies, it is convincingly shown that, in the mixed conformational pool, the heterogeneous backbone of the hybrid peptides preferentially adopt periodic 8-membered (pseudo γ-turn)/7-membered (inverse γ-turn) hydrogen bonds in both polar and non-polar solvent media. It is observed that the stereochemistry and local conformational preference of the ß-amino acid building blocks have a profound influence on accessing the specific secondary fold. These findings may be of significant relevance for the development of molecular scaffolds that facilitate desired positioning of functional side-chains.
Asunto(s)
Péptidos/química , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de ProteínaRESUMEN
An efficient and practical total synthesis of marine cyclic tetrapeptide, natural product azumamide E (1) is achieved via high-yielding reactions. The strategy also allowed us to synthesize the azumamide E-SAA (sugar amino acid) analogue (2), whose solution-phase NMR and biological activity studies were also carried out.