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Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

ABSTRACT: Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

SUBMITTER: Poreba M

PROVIDER: S-EPMC3962583 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.

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Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction.

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A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids.

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Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time.

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Synthesis of Enantiopure Unnatural Amino Acids by Metallaphotoredox Catalysis.

Project description:We describe herein a two-step process for the conversion of serine to a wide array of optically pure unnatural amino acids. This method utilizes a photocatalytic cross-electrophile coupling between a bromoalkyl intermediate and a diverse set of aryl halides to produce artificial analogues of phenylalanine, tryptophan, and histidine. The reaction is tolerant of a broad range of functionalities and can be leveraged toward the scalable synthesis of valuable pharmaceutical scaffolds via flow technology.

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Enzymatic aminoacylation of tRNA with unnatural amino acids.

Project description:The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only approximately 75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified peptides. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many beta-amino acids, N-methyl amino acids, and alpha,alpha-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified peptides.

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Study and design of amino acid-based radical enzymes using unnatural amino acids.

Project description:Radical enzymes harness the power of reactive radical species by placing them in a protein scaffold, and they are capable of catalysing many important reactions. New native radical enzymes, especially those with amino acid-based radicals, in the category of non-heme iron enzymes (including ribonucleotide reductases), heme enzymes, copper enzymes, and FAD-radical enzymes have been discovered and characterized. We discussed recent research efforts to discover new native amino acid-based radical enzymes, and to study the roles of radicals in processes such as enzyme catalysis and electron transfer. Furthermore, design of radical enzymes in a small and simple scaffold not only allows us to study the radical in a well-controlled system and test our understanding of the native enzymes, but also allows us to create powerful enzymes. In the study and design of amino acid-based radical enzymes, the use of unnatural amino acids allows precise control of pKa values and reduction potentials of the residue, as well as probing the location of the radical through spectroscopic methods, making it a powerful research tool. Our understanding of amino acid-based radical enzymes will allow us to tailor them to create powerful catalysts and better therapeutics.

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| S-EPMC4299457 | biostudies-literature

Visible-light photoredox synthesis of unnatural chiral α-amino acids.

Project description:Unnatural chiral α-amino acids are widely used in fields of organic chemistry, biochemistry and medicinal chemistry, and their synthesis has attracted extensive attention. Although the asymmetric synthesis provides some efficient protocols, noble and elaborate catalysts, ligands and additives are usually required which leads to high cost. Distinctly, it is attractive to make unnatural chiral α-amino acids from readily available natural α-amino acids through keeping of the existing chiral α-carbon. However, it is a great challenge to construct them under mild conditions. In this paper, 83 unnatural chiral α-amino acids were prepared at room temperature under visible-light assistance. The protocol uses two readily available genetically coded proteinogenic amino acids, L-aspartic acid and glutamic acid derivatives as the chiral sources and radical precursors, olefins, alkynyl and alkenyl sulfones, and 2-isocyanobiphenyl as the radical acceptors, and various unnatural chiral α-amino acids were prepared in good to excellent yields. The simple protocol, mild conditions, fast reactions, and high efficiency make the method an important strategy for synthesis of diverse unnatural chiral α-amino acids.

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Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.

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