Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids.

ABSTRACT: The importance of bioconjugates within the field of chemistry drives the need for novel methodologies for their preparation. Well-defined and stable bioconjugates are easily accessible via the utilization of unnatural amino acids (UAAs). As such, we have synthesized and incorporated two new UAAs into green fluorescent protein, and optimized a novel Cadiot-Chodkiewicz bioconjugation, effectively expanding the toolbox of chemical reactions that can be employed in the preparation of bioconjugates.

SUBMITTER: Maza JC

PROVIDER: S-EPMC5461652 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids.

Maza Johnathan C JC Nimmo Zachary M ZM Young Douglas D DD

Chemical communications (Cambridge, England) 20160101 1

The importance of bioconjugates within the field of chemistry drives the need for novel methodologies for their preparation. Well-defined and stable bioconjugates are easily accessible via the utilization of unnatural amino acids (UAAs). As such, we have synthesized and incorporated two new UAAs into green fluorescent protein, and optimized a novel Cadiot-Chodkiewicz bioconjugation, effectively expanding the toolbox of chemical reactions that can be employed in the preparation of bioconjugates. ...[more]

PMID: 26499242

Similar Datasets

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

Project description:In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as ?-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.

| S-EPMC7582959 | biostudies-literature

Visible-light-mediated catalyst-free synthesis of unnatural α-amino acids and peptide macrocycles.

Project description:The visible light induced, photocatalysts or photoabsorbing EDA complexes mediated cleavage of pyridinium C-N bond were reported in the past years. Here, we report an ionic compound promote homolytic cleavage of pyridinium C-N bond by exploiting the photonic energy from visible light. This finding is successfully applied in deaminative hydroalkylation of a series of alkenes including naturally occurring dehydroalanine, which provides an efficient way to prepare β-alkyl substituted unnatural amino acids under mild and photocatalyst-free conditions. Importantly, by using this protocol, the deaminative cyclization of peptide backbone N-terminals is realized. Furthermore, the use of Et3N or PPh3 as reductants and H2O as hydrogen atom source is a practical advantage. We anticipate that our protocol will be useful in peptide synthesis and modern peptide drug discovery.

| S-EPMC8617070 | biostudies-literature

Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings.

Project description:Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells.

| S-EPMC3947478 | biostudies-literature

Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations.

Project description:Employing selenocysteine-containing protein fragments to form the amide bond between respective protein fragments significantly extends the current capabilities of the widely used protein engineering method, expressed protein ligation. Selenocysteine-mediated ligation is noteworthy for its high yield and efficiency. However, it has so far been restricted to solid-phase synthesized seleno-peptides and thus constrained by where the selenocysteine can be positioned. Here we employ heterologously expressed seleno-fragments to overcome the placement and size restrictions in selenocysteine-mediated chemical ligation. Following ligation, the selenocysteine can be deselenized into an alanine or serine, resulting in nonselenoproteins. This greatly extends the flexibility in selecting the conjugation site in expressed protein ligations with no influence on native cysteines. Furthermore, the selenocysteine can be used to selectively introduce site-specific protein modifications. Therefore, selenocysteine-mediated expressed protein ligation simplifies incorporation of post-translational modifications into the protein scaffold.

| S-EPMC5824972 | biostudies-literature

Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair.

Project description:As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.

| S-EPMC3690937 | biostudies-literature

Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair.

Project description:We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs to be site-specifically incorporated into proteins in yeast.

| S-EPMC2956376 | biostudies-literature

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.

| S-EPMC9162430 | biostudies-literature

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.

| S-EPMC1450175 | biostudies-other

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.

| S-EPMC10246556 | biostudies-literature

Synthesis of bispecific antibodies using genetically encoded unnatural amino acids.

Project description:Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.

| S-EPMC4299457 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data