Hydroxylation Of Ethylbenzene Research Articles

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in <i>Escherichia coli</i>

Rational design of an artificial ethylbenzene hydroxylase using a molecular dynamics simulation to enhance enantioselectivity

Abstract Mutations of myoglobin reconstituted with Mn porphycene (rMb) were investigated to enhance the enantioselectivity for hydroxylation of ethylbenzene. The 21 mutants of rMb predicted by models using molecular dynamics simulation were prepared. Several rMb mutants enhance the enantiomeric excess (ee) values up to 69% and 57% for (S)- and (R)-1-phenylethanols, respectively, compared with wild-type rMb (17% ee for (S)-1-phenylethanol). Furthermore, the crystal structures demonstrate slightly expanded spaces to support the substrate binding behavior indicated in the simulation.

C(sp3)-H hydroxylation of Broad-Spectrum Alkanes Catalyzed by an Artificial P450 Peroxygenase Driven by ω-Pyridyl Fatty Acyl Amino Acids

Selective hydroxylation of C(sp3)–H bonds is a long-standing challenge in the organic synthesis field. We herein report an artificial P450 peroxygenase system facilitated by ω-pyridyl fatty acyl amino acids that can hydroxylate broad-spectrum alkanes, including linear and cyclic alkanes and benzylic and allylic alkane chains. Compared with the previously reported dual-functional small molecule (DFSM) with imidazole as a catalytic group (Im-DFSM), pyridyl DFSM (Pyd-DFSM) produced substantially higher total turnover numbers (TTNs) for the hydroxylation of medium-chain alkanes and cycloalkanes (C6–C8), e.g., 12.0-fold and 17.1-fold increased TTNs of P450 mutant F87A/T268V for heptane and cycloheptane, respectively. In addition, Pyd-DFSM altered the enantioselectivity of benzylic hydroxylation, e.g., it almost inverted the configuration observed in the benzylic hydroxylation of ethylbenzene and tetralin. More importantly, Pyd-DFSM showed excellent regioselectivity for allylic hydroxylation over epoxidation of terminal alkenes and cycloalkenes, e.g., up to 94% allylic hydroxylation of 1-hexene was observed. This work significantly enhances the catalytic toolbox of the DFSM-facilitated P450 peroxygenase system; it not only substantially expands the chemical space of P450 enzymes, but also provides an alternative solution for the selective hydroxylation of C(sp3)–H bonds.

Molecular Dynamics of Iron Porphyrin-Catalyzed C-H Hydroxylation of Ethylbenzene.

Quasi-classical molecular dynamics (MD) simulations were carried out to study the mechanism of iron porphyrin-catalyzed hydroxylation of ethylbenzene. The hydrogen atom abstraction from ethylbenzene by iron-oxo species is the rate-determining step, which generates the radical pair of iron-hydroxo species and the benzylic radical. In the subsequent radical rebound step, the iron-hydroxo species and benzylic radical recombine to form the hydroxylated product, which is barrierless on the doublet energy surface. In the gas-phase quasi-classical MD study on the doublet energy surface, 45% of the reactive trajectories lead directly to the hydroxylated product, and this increases to 56% in implicit solvent model simulations. The percentage of reactive trajectories leading to the separated radical pair is 98-100% on high-spin (quartet/sextet) energy surfaces. The low-spin state reactivity dominates in the hydroxylation of ethylbenzene, which is dynamically both concerted and stepwise, since the time gap between C-H bond cleavage and C-O bond formation ranges from 41 to 619 fs. By contrast, the high-spin state catalysis is an energetically stepwise process, which has a negligible contribution to the formation of hydroxylation products.

A novel unspecific peroxygenase from Agaricus bisporus var. bisporus for biocatalytic oxyfunctionalisation reactions

Unspecific peroxygenases (UPOs) represent an emerging class of catalysts for the selective oxyfunctionalisation of CH- and C = C groups. Until now, only a few UPOs have been characterised. In this study, we report a new peroxygenase identified from the Unspecific Peroxygenase Database. The UPO from Agaricus bisporus var. bisporus (AbvbUPO) has been heterologously expressed in Aspergillus niger and initially characterised with respect to its basic biochemical features. Furthermore, its catalytic properties were evaluated with enzymatic cascade reactions of choline oxidase (AnChOx) and AbvbUPO, which the AnChOx provided H2O2 necessary via reductive activation of oxygen in situ. Three types of oxyfunctionalizations, such as hydroxylation of ethylbenzene, epoxidations of styrene and cyclohexene, sulfoxidations of methyl phenyl sulfide and phenyl vinyl sulfide, were successfully achieved. We also investigated the activity of AbvbUPO on fatty acids in some more detail. The experimental results show that Under the above conditions, AbvbUPO had the higher activity for cyclohexene epoxidation and sulfonation of sulfide substrates. The concentration of epoxy cyclohexane was 2.91 mM, and the concentration of methyl phenyl sulfoxide was 3.69 mM. The regioselectivity of AbvbUPO was ω-1 bonds position of linear saturated fatty acid. All in all, AbvbUPO exhibits some interesting differences which may put the basis for further understanding of the factors determining peroxygenase selectivity.

Peroxygenase-Driven Ethylbenzene Hydroxylation in a Rotating Bed Reactor

Peroxygenase-Driven Ethylbenzene Hydroxylation in a Rotating Bed Reactor

Glucose Induces Heme Leakage and Suppresses H2O2 Uptake of Chloroperoxidase in the Asymmetric Hydroxylation of Ethylbenzene

AbstractAs a basic feedstock for both natural life and chemical production, glucose is converted into various chemicals upon the help of peroxidase cascades composed of glucose oxidase (GOx) and peroxidases. However, little is known about the glucose effect on the structure and activity of peroxidases in a non‐natural environment. We report herein that glucose deactivates chloroperoxidase (CPO) used in the asymmetric hydroxylation of ethylbenzene into (R)‐1‐phenylethanol. Through both experiment and molecular simulation, we discover that glucose deactivates CPO through hydrophobic interaction with heme and hydrogen bonding with Glu183. These interactions lead to heme leakage and the reduction of H2O2 uptake thereby undermining the CPO activity. The mechanism appears generically applicable to other hemeproteins such as horseradish peroxidase, hemoglobin, and myoglobin. These findings may open new avenues for the design of peroxidase cascades for applications ranging from better diagnosis of diabetes to green synthesis of chemicals by direct functionalization of C−H bonds.

Insight into the methylene C–H bond cleavage of ethylbenzene during ethylbenzene hydroxylation using EBDH as a catalyst, a DFT study

The hydroxylation of ethylbenzene to ( S)-1-phenylethanol with the help of ethylbenzene dehydrogenase (EBDH) is a stereospecific catalytic reaction. This hydroxylation process involves the C–H bond cleavage of methylene part of ethylbenzene and transfer of its hydrogen to the oxygen atom attached with the metal at the active site of EBDH as a first step, which leads to the formation of an intermediate. The second step involves the transfer of OH from the active-site metal back to the carbon of intermediate, resulting in the formation of ( S)-1-phenylethanol. This C–H bond cleavage could be homolytic or heterolytic and directly affect the reaction mechanism of ethylbenzene hydroxylation. In this article, density functional theory studies were performed on the ethylbenzene-bound EBDH active-site model complexes to investigate the impact of the C–H bond cleavage of methylene part of ethylbenzene on the reaction mechanism of ethylbenzene hydroxylation. For this, different protonation states and participation of amino acid residues near the Mo center of EBDH were considered. Models with protonation of His192, Lys450, and Asp223 and model without protonation were investigated for comparison. Computed relative energies indicate that the overall lowest energy barrier pathway results when ionic (heterolytic) and radical (homolytic) pathways are combined.

A Core‐Shell Cascade of Chloroperoxidase and Gold Nanoclusters for Asymmetric Hydroxylation of Ethylbenzene

AbstractDirect functionalization of C−H bonds catalyzed by chloroperoxidase (CPO) is featured by using H2O2 as both oxygen and electron donor. The catalytic process circumvents expensive electron transporters such as NAD(P)H, making it advantageous for green production of fine chemicals with complex structures. In situ generation and collaborative supplement of H2O2 is strongly desired for an improved productivity and enzyme stability. The present study describes a novel CPO cascade for asymmetric hydroxylation of ethylbenzene yielding (R)‐1‐phenylethanol, in which H2O2 is generated by oxidation of monosaccharides catalyzed by gold nanoclusters (AuNCs). A core‐shell structure of CPO−AuNCs cascade was prepared by using hollow mesoporous silica microspheres (HMSMs) as the support. AuNCs were immobilized on the external surface of HMSMs while CPOs were encapsulated inside the microspheres. Different monosaccharides were examined for H2O2 production as a function of the particle size. It was found that galactose exhibits the highest productivity. The high compatibility of CPO and AuNCs against temperature and pH is advantageous in catalyzing a wide spectrum of reactions for C−H functionalization.

Deracemization of 1-phenylethanols in a one-pot process combining Mn-driven oxidation with enzymatic reduction utilizing a compartmentalization technique.

Racemic 1-phenylethanols were converted into enantiopure (R)-1-phenylethanols via a chemoenzymatic process in which manganese oxide driven oxidation was coupled with enzymatic biotransformation by compartmentalization of the reactions, although the two reactions conducted under mixed conditions are not compatible due to enzyme deactivation by Mn ions. Achiral 1-phenylethanol is oxidized to produce acetophenone in the interior chamber of a polydimethylsiloxane thimble. The acetophenone passes through the membrane into the exterior chamber where enantioselective biotransformation takes place to produce (R)-1-phenylethanol with an enantioselectivity of >99% ee and with 96% yield. The developed sequential reaction could be applied to the deracemization of a wide range of methyl- and chloro-substituted 1-phenylethanols (up to 93%, >99% ee). In addition, this method was applied to the selective hydroxylation of ethylbenzene to afford chiral 1-phenylethanol.

Pilot-Scale Production of Peroxygenase from Agrocybe aegerita.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways*.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible‐light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN‐OA‐m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo‐chemo‐enzymatic cascades that give either the (S)‐ or the (R)‐enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN‐OA‐m led to the enantioselective hydroxylation of ethylbenzene to (R)‐1‐phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)‐1‐phenylethanol (93 % ee).

Solar-Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions.

Inspired by natural photosynthesis, biocatalytic photoelectrochemical (PEC) platforms are gaining prominence for the conversion of solar energy into useful chemicals by combining redox biocatalysis and photoelectrocatalysis. Herein, we report a dual biocatalytic PEC platform consisting of a molybdenum (Mo)-doped BiVO4 (Mo:BiVO4 ) photoanode and an inverse opal ITO (IO-ITO) cathode that gives rise to the coupling of peroxygenase and ene-reductase-mediated catalysis, respectively. In the PEC cell, the photoexcited electrons generated from the Mo:BiVO4 are transferred to the IO-ITO and regenerate reduced flavin mononucleotides to drive ene-reductase-catalyzed trans-hydrogenation of ketoisophrone to (R)-levodione. Meanwhile, the photoactivated Mo:BiVO4 evolves H2 O2 in situ via a two-electron water-oxidation process with the aid of an applied bias, which simultaneously supplies peroxygenases to drive selective hydroxylation of ethylbenzene into enantiopure (R)-1-phenyl-1-hydroxyethane. Thus, the deliberate integration of PEC systems with redox biocatalytic reactions can simultaneously produce valuable chemicals on both electrodes using solar-powered electrons and water.

Solar‐Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions

AbstractInspired by natural photosynthesis, biocatalytic photoelectrochemical (PEC) platforms are gaining prominence for the conversion of solar energy into useful chemicals by combining redox biocatalysis and photoelectrocatalysis. Herein, we report a dual biocatalytic PEC platform consisting of a molybdenum (Mo)‐doped BiVO4 (Mo:BiVO4) photoanode and an inverse opal ITO (IO‐ITO) cathode that gives rise to the coupling of peroxygenase and ene‐reductase‐mediated catalysis, respectively. In the PEC cell, the photoexcited electrons generated from the Mo:BiVO4 are transferred to the IO‐ITO and regenerate reduced flavin mononucleotides to drive ene‐reductase‐catalyzed trans‐hydrogenation of ketoisophrone to (R)‐levodione. Meanwhile, the photoactivated Mo:BiVO4 evolves H2O2 in situ via a two‐electron water‐oxidation process with the aid of an applied bias, which simultaneously supplies peroxygenases to drive selective hydroxylation of ethylbenzene into enantiopure (R)‐1‐phenyl‐1‐hydroxyethane. Thus, the deliberate integration of PEC systems with redox biocatalytic reactions can simultaneously produce valuable chemicals on both electrodes using solar‐powered electrons and water.

Computational Exploration of Chiral Iron Porphyrin-Catalyzed Asymmetric Hydroxylation of Ethylbenzene Where Stereoselectivity Arises from π-π Stacking Interaction.

The mechanism and origins of stereoselectivity of chiral iron porphyrin-catalyzed asymmetric hydroxylation of ethylbenzene were explored with density functional theory. The hydrogen atom abstraction is the rate- and stereoselectivity-determining step. In good agreement with experimental results, the formation of the (R)-1-phenylethanol product is found to be the most favorable pathway. The transition state of hydrogen atom abstraction which leads to the (S)-1-phenylethanol product is unfavorable by 1.7 kcal/mol compared to the corresponding transition state which leads to the (R)-1-phenylethanol product. Enantioselectivity arises from an attractive π-π stacking interaction between the phenyl group of ethylbenzene substrate and the naphthyl group of the porphyrin ligand.

Photoenzymatic Hydroxylation of Ethylbenzene Catalyzed by Unspecific Peroxygenase: Origin of Enzyme Inactivation and the Impact of Light Intensity and Temperature

AbstractPhotoenzymatic cascades can be used for selective oxygenation of C−H‐Bonds under mild conditions circumventing the hydrogen peroxide mediated peroxygenase inactivation via in situ H2O2 generation. Here, we report the “on demand” production of hydrogen peroxide via methanol assisted reduction of molecular oxygen using UV‐illuminated titanium dioxide (Aeroxide P25) combined with the enantioselective hydroxylation of ethylbenzene to (R)‐1‐phenylethanole catalyzed by the Unspecific Peroxygenase from Agrocybe Aegerita. For the application of the system it is important to understand the influence of the reaction parameters to be able to optimize the system. Therefore, we systematically investigated product formation and enzyme inactivation as well as ROS formation (H2O2, .OH and .O2−) applying different light intensities and temperatures. As a result, Turnover Numbers up to 220 000, photonic efficiencies up to 13.6 % and production rates up to 0.9 mM h−1 were achieved.

Catalytic and stoichiometric C[sbnd]H oxidation of benzylalcohols and hydrocarbons mediated by nonheme oxoiron(IV) complex with chiral tetrapyridyl ligand

Bioinspired chiral iron(II) complex [((R)-(−)-N4Py*)FeII(CH3CN)]2+ (1) (N4Py* = N,N-bis(2-pyridylmethyl)-1,2-di(2-pyridyl)ethylamine) has been shown to efficiently catalyze the benzylic CH oxidation of ethylbenzene with tert-butyl hydroperoxide (TBHP), H2O2, and meta-chloroperoxybenzoic acid (mCPBA) resulting in enantiomerically enriched 1-phenylethanol up to 12.5% ee and the corresponding acetophenone, where the [FeIV(N4Py*)(O)]2+ (2) intermediate has been detected by UV/Vis spectrometry. The stoichiometric oxidation of benzyl alcohol and various hydrocarbon derivatives including the asymmetric hydroxylation of ethylbenzene with 2 has also been investigated. Detailed kinetic, and mechanistic studies (kinetic isotop effect (KIE) of 31 and 38, and Hammett correlation with ρ = −0.32 and −0.98 for PhCH2OH and PhCH3, respectively, and the linear correlation between the normalized bimolecular reaction rates and bond dissociation energies (BDECH)) lead to the conclusion that the rate-determining step in these reactions above involves hydrogen-atom transfer between the substrate and the Fe(IV)-oxo species. The stoichiometric 2-mediated hydroxylation of ethylbenzene affords 1-phenylethanol in up to 33% ee, suggesting clear evidence for the involvement of the oxoiron(IV) species in the enantioselective step. The moderate enantioselectivity may be explained by the epimerization of the long-lived substrate radical before the rebound step (non-rebound mechanism, where kep > kreb). The kinetic resolution of the resulting chiral alcohol due to its metal-based overoxidation process into acetophenone in the catalytic metal-based ethylbenzene oxidation can be excluded.

Peroxygenase-Catalyzed Hydroxylation of Ethylbenzene

Peroxygenase-Catalyzed Hydroxylation of Ethylbenzene

Dual-Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase.

We report a unique strategy for the development of a H2 O2 -dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid-base catalyst in the activation of H2 O2 . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H2 O2 system previously reported. This work provides the first example of the activation of the normally H2 O2 -inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.