Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds.

ABSTRACT: RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para- to the indole nitrogen were evolved by directly screening for altered selectivity on deuterium-substituted probe substrates using mass spectrometry. This systematic approach allowed for rapid accumulation of beneficial mutations using simple adaptive walks and should prove generally useful for altering and optimizing the selectivity of C-H functionalization catalysts. Analysis of the beneficial mutations showed that structure-guided selection of active site residues for targeted mutagenesis can be complicated either by activity/selectivity tradeoffs that reduce the possibility of detecting such mutations or by epistatic effects that actually eliminate the benefits of a mutation in certain contexts. As a corollary to this finding, the precise manner in which the beneficial mutations identified led to the observed changes in RebH selectivity is not clear. Docking simulations suggest that tryptamine binds to these variants as tryptophan does to native halogenases, but structural studies will be required to confirm these models and shed light on how particular mutations impact tryptamine binding. Similar directed evolution efforts on other enzymes or artificial metalloenzymes could enable a wide range of C-H functionalization reactions.

SUBMITTER: Andorfer MC

PROVIDER: S-EPMC4917012 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Directed evolution of RebH for site-selective halogenation of large biologically active molecules.

Project description:We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.

| S-EPMC4506780 | biostudies-literature

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

Project description:We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3-LR, with a Topt over 5?°C higher than that of wild-type, and 3-LSR, with a Tm 18?°C higher than that of wild-type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non-natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.

| S-EPMC4124618 | biostudies-literature

Regioselective arene halogenation using the FAD-dependent halogenase RebH.

Project description: Not available

| S-EPMC3824154 | biostudies-literature

RebH-Catalyzed Indole and Azaindole Halogenation Utilizing Co-Purified E. Coli Reductase

Project description:IP-mass spec to determine proteins binding to RebH enzyme expressed in E. coli.

2022-10-30 | MSV000090619 | MassIVE

Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds.

Project description:Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure-guided semi-rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.

| S-EPMC8518859 | biostudies-literature

Metalloenzyme-Inspired Ce-MOF Catalyst for Oxidative Halogenation Reactions.

Project description:The structure of UiO-66(Ce) is formed by CeO2-x defective nanoclusters connected by terephthalate ligands. The initial presence of accessible Ce3+ sites in the as-synthesized UiO-66(Ce) has been determined by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR)-CO analyses. Moreover, linear scan voltammetric measurements reveal a reversible Ce4+/Ce3+ interconversion within the UiO-66(Ce) material, while nanocrystalline ceria shows an irreversible voltammetric response. This suggests that terephthalic acid ligands facilitate charge transfer between subnanometric metallic nodes, explaining the higher oxidase-like activity of UiO-66(Ce) compared to nanoceria for the mild oxidation of organic dyes under aerobic conditions. Based on these results, we propose the use of Ce-based metal-organic frameworks (MOFs) as efficient catalysts for the halogenation of activated arenes, as 1,3,5-trimethoxybenzene (TMB), using oxygen as a green oxidant. Kinetic studies demonstrate that UiO-66(Ce) is at least three times more active than nanoceria under the same reaction conditions. In addition, the UiO-66(Ce) catalyst shows an excellent stability and can be reused after proper washing treatments. Finally, a general mechanism for the oxidative halogenation reaction is proposed when using Ce-MOF as a catalyst, which mimics the mechanistic pathway described for metalloenzymes. The superb control in the generation of subnanometric CeO2-x defective clusters connected by adequate organic ligands in MOFs offers exciting opportunities in the design of Ce-based redox catalysts.

| S-EPMC9131423 | biostudies-literature

Non-Heme-Iron-Mediated Selective Halogenation of Unactivated Carbon-Hydrogen Bonds.

Project description:Oxidation of the iron(II) precursor [(L1 )FeII Cl2 ], where L1 is a tetradentate bispidine, with soluble iodosylbenzene (s PhIO) leads to the extremely reactive ferryl oxidant [(L1 )(Cl)FeIV =O]+ with a cis disposition of the chlorido and oxido coligands, as observed in non-heme halogenase enzymes. Experimental data indicate that, with cyclohexane as substrate, there is selective formation of chlorocyclohexane, the halogenation being initiated by C-H abstraction and the result of a rebound of the ensuing radical to an iron-bound Cl- . The time-resolved formation of the halogenation product indicates that this primarily results from s PhIO oxidation of an initially formed oxido-bridged diiron(III) resting state. The high yield of up to >70 % (stoichiometric reaction) as well as the differing reactivities of free Fe2+ and Fe3+ in comparison with [(L1 )FeII Cl2 ] indicate a high complex stability of the bispidine-iron complexes. DFT analysis shows that, due to a large driving force and small triplet-quintet gap, [(L1 )(Cl)FeIV =O]+ is the most reactive small-molecule halogenase model, that the FeIII /radical rebound intermediate has a relatively long lifetime (as supported by experimentally observed cage escape), and that this intermediate has, as observed experimentally, a lower energy barrier to the halogenation than the hydroxylation product; this is shown to primarily be due to steric effects.

| S-EPMC9300152 | biostudies-literature

A simple base-mediated halogenation of acidic sp2 C-H bonds under noncryogenic conditions.

Project description:A new method has been developed for in situ halogenation of acidic sp(2) carbon-hydrogen bonds in heterocycles and electron-deficient arenes. Either selective monohalogenation or one-step exhaustive polyhalogenation is possible for substrates possessing several C-H bonds that are flanked by electron-withdrawing groups. For the most acidic arenes, such as pentafluorobenzene, K(3)PO(4) base can be employed instead of BuLi for metalation/halogenation sequences.

| S-EPMC2635093 | biostudies-literature

Electrochemical Oxidative Clean Halogenation Using HX/NaX with Hydrogen Evolution.

Project description:Organic halides (R-X) are prevalent structural motifs in pharmaceutical molecules and key building blocks for the synthesis of fine chemicals. Although a number of routes are available in the literature for the synthesis of organic halides, these methods often require stoichiometric additives or oxidants, metal catalysts, leaving or directing groups, or toxic halogenating agents. In addition, the necessity of employing different, often tailor-made, catalytic systems for each type of substrate also limits the applicability of these methods. Herein, we report a clean halogenation by electrochemical oxidation with NaX/HX. A series of organic halides were prepared under metal catalyst- and exogenous-oxidant-free reaction conditions. It is worth noting that this reaction has a broad substrate scope; various heteroarenes, arenes, alkenes, alkynes, and even aliphatic hydrocarbons could be applied. Most importantly, the reaction could also be performed on a 200-mmol scale with the same efficiency (86%, 50.9 g pure product).

| S-EPMC6365813 | biostudies-other

A visible-light-promoted radical reaction system for azidation and halogenation of tertiary aliphatic C-H bonds.

Project description:A highly tunable radical-mediated reaction system for the functionalization of tertiary aliphatic C-H bonds was developed. Reactions of various substrates with the Zhdankin azidoiodane reagent 1, Ru(bpy)3Cl2, and visible light irradiation at room temperature gave C-H azidated or halogenated products in an easily controllable fashion. These reactions are efficient, selective, and compatible with complex substrates. They provide a potentially valuable tool for selectively labeling tertiary C-H bonds of organic and biomolecules with tags of varied chemical and biophysical properties for comparative functional studies.

| S-EPMC5477033 | 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