Enantioselective Epoxidation Of Styrene Research Articles

Emerging Strategies for Modifying Cytochrome P450 Monooxygenases into Peroxizymes.

ConspectusCytochrome P450 monooxygenase is a versatile oxidizing enzyme with great potential in synthetic chemistry and biology. However, the dependence of its catalytic function on the nicotinamide cofactor NAD(P)H and redox partner proteins limits the practical catalytic application of P450 in vitro. An alternative to expensive cofactors is low-cost H2O2, which can be used directly to exploit the catalytic potential of P450s. However, the peroxide shunt pathway is generally inefficient at driving P450 catalysis compared to normal NAD(P)H-dependent activity. Over the last few decades, the scientific community has made continuous efforts to use directed evolution or site-directed mutagenesis to modify P450 monooxygenases into their peroxizyme modes─peroxygenase and peroxidase. Despite significant progress, obtaining efficient P450 peroxizymes remains a huge challenge. Here, we summarize our efforts to modulate peroxizyme activity in P450 monooxygenases and exploit their catalytic applications in challenging selective C-H oxidation, oxygenation, and oxyfunctionalization over the past seven years. We first developed a dual-functional small molecule (DFSM) strategy for transforming P450BM3 monooxygenase into peroxygenase. In this strategy, the typical DFSM, such as N-(ω-imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe), binds to the P450BM3 protein with an anchoring group at one end and plays a general acid-base catalytic role in the activation of H2O2 with an imidazolyl group at the other end. Compared with the O-O homolysis mechanism in the absence of DFSM, the addition of DFSM efficiently enables the heterolytic O-O cleavage of the adduct Fe-O-OH, thus being favored for the formation of active species compound I, which has been demonstrated by combining crystallographic and theoretical calculations. Furthermore, protein engineering showed the unique catalytic performance of DFSM-facilitated P450 peroxygenase for the highly difficult selective oxidation of C-H bonds. This catalytic performance was demonstrated during the chemoselective hydroxylation of gaseous alkanes, regioselective O-demethylation of aryl ethers, highly (R)-enantioselective epoxidation of styrene, and regio- and enantiomerically diverse hydroxylation of alkylbenzenes. Second, we demonstrated that DFSM-facilitated P450BM3 peroxygenase could be effectively switched to an efficient peroxidase mode through mechanism-guided protein engineering of redox-sensitive residues. Utilizing the peroxidase function of P450 enabled the direct nitration of unsaturated hydrocarbons including phenols, aromatic amines, and styrene derivatives, which was not only the P450-catalyzed direct nitration of phenols and aromatic amines for the first time but also the first example of the direct biological nitration of olefins. Finally, we report an H2O2 tunnel engineering strategy to enable peroxygenase activity in several different P450 monooxygenases for the first time, providing a general approach for accessing engineered P450 peroxygenases. In this Account, we highlight the emerging strategies we have developed for producing practical P450 peroxizyme biocatalysts. Although the DFSM strategy is primarily applied to P450BM3 to date, both strategies of redox-sensitive residue engineering and H2O2 tunnel engineering show great potential to extend to other P450s. These strategies have expanded the scope of applications of P450 chemistry and catalysis. Additionally, they provide a unique solution to the challenging selective oxidation of inert C-H bonds in synthetic chemistry.

Enabling highly (R)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases.

Unlike the excellent (S)-enantioselective epoxidation of styrene performed by natural styrene monooxygenases (ee > 99%), the (R)-enantioselective epoxidation of styrene has not yet achieved a comparable efficiency using natural or engineered oxidative enzymes. This report describes the H2O2-dependent (R)-enantioselective epoxidation of unfunctionalized styrene and its derivatives by site-mutated variants of a unique non-natural P450BM3 peroxygenase, working in tandem with a dual-functional small molecule (DFSM). The observed (R)-enantiomeric excess (ee) of styrene epoxidation is up to 99% with a turnover number (TON) of 918 by the best enantioselective mutant F87A/T268I/L181Q, while the best active mutant F87A/T268I/V78A/A184L (with 98% ee) gave a catalytic TON of 4350, representing the best activity of a P450 peroxygenase towards styrene epoxidation to date. Following this approach, a set of styrene derivatives, such as o-, m-, p-chlorostyrenes and fluorostyrenes, could also be epoxidized with modest to very good TONs (362–3480) and high (R)-enantioselectivities (95–99% ee). The semi-preparative scale synthesis of (R)-styrene oxide performed at 0 °C with high conversion, maintaining enantioselectivity, and moderate isolated yields, further suggests the potential application of the current P450 enzymatic system in styrene epoxidation. This study indicates that the synergistic use of protein engineering and an exogenous DFSM constitutes an efficient strategy to control the enantioselectivity of styrene epoxidation, thus substantially expanding the chemical scope of P450 enzymes as useful bio-oxidative catalysts.

Electrochemical Reduction of FAD for NSMOA‐Catalyzed Epoxidation Reactions

Styrene monooxygenase (SMO) is a two‐component flavoprotein composed of an NADH‐dependent flavin‐reductase (SMOB) and FAD‐dependent epoxidase (NSMOA). The SMO‐catalyzed enantioselective epoxidation of styrene has potential applications in green‐chemical aqueous phase synthesis and environmental remediation. In the present work, we investigate the possibility of eliminating the NADH‐dependence of this two‐component biocatalyst by developing an electrochemical system that can be substituted for SMOB as the source of reduced FAD as the rate‐limiting step in the styrene epoxidation reaction.In this work, a miniature spectroelectrochemical cell was constructed and interfaced with a potentiostat that enabled the study of electrode‐mediated flavin‐reduction reactions by cyclic voltammetry and diode array‐based UV‐vis spectroscopy under anaerobic and aerobic conditions. Different results were obtained depending on the composition of the working electrode in terms of the kinetics of FAD desorption from the electrode surface and the applied electrical potential required to achieve FAD reduction. Glassy carbon and graphene‐based working electrodes are observed to readily establish and remain in electrical communication with FAD, but the kinetics of FAD reduction in bulk solution is limited by the slow kinetics of FAD desorption from the surface of these working electrodes. Under aerobic conditions, oxygen diffuses within and reacts with reduced FAD adsorbed to the electrode surface in a reaction which is rapid compared with the kinetics of the desorption of reduced FAD. The implementation of more hydrophilic working electrodes prepared as composites of graphene and polyethylene glycol and the inclusion of electron carriers with oxidation reduction‐potentials in the range of FAD significantly improves the rate FAD desorption and reduction in bulk solution where the rapid kinetics of association of reduced FAD to the epoxidase (NSMOA) can efficiently out compete undesirable side reactions of reduced FAD with dissolved oxygen. In this work, we address the central challenges associated with the electrochemical reduction preparation and transmission of reduced FAD to styrene monooxygenase and report how this can be achieved efficiently by using a combination of cyclic voltammetry, electron mediators and an electrode material with rapid FAD desorption characteristics.Support or Funding InformationThis research is founded by NIH MBRS‐RISE (R25‐GM059298).Cyclic voltammetry of FAD at the surface of a glassy carbon electrode and saturation plot of the flavin interaction at the electrode surface.Figure 1Absorbance spectra recording the reduction of FAD (Left) and consumption of styrene (right) by UV‐Vis spectroscopy.Figure 2

The styrene monooxygenase system.

Styrene monooxygenases are soluble two-component flavoproteins that catalyze the NADH and FAD-dependent enantioselective epoxidation of styrene to styrene oxide in the aqueous phase. These enzymes present interesting mechanistic features and potential as catalysts in biotechnological applications ranging from green chemical synthesis to bioremediation. This chapter presents approaches for the expression of the reductase (SMOB, StyB) and epoxidase (SMOA, StyA) components of SMO from pET-vectors as native or N-terminally histidine-tagged proteins in commercial strains of E. coli. The two-component structure of SMO and hydrophobic nature of styrene substrate requires some special consideration in evaluating the mechanism of this enzyme. The modular composition of the enzyme allows the flavin-reduction reaction of SMOB and styrene epoxidation reaction of SMOA to be evaluated both independently and as a composite catalytic system. The freedom to independently study the reductase and epoxidase components of SMO significantly simplifies studies of equilibrium-binding and the coupling of the free energy of ligand binding to the electrochemical potential of bound FAD. In this chapter, methods of steady-state and pre-steady-state kinetic assay, experimental approaches to equilibrium-binding reactions of flavin and substrate, and determination of the electrochemical midpoint potential of FAD bound to the reductase and epoxidase components of SMO are presented. This presentation focuses on approaches that have been successfully used in the study of the wild-type styrene monooxygenase system recovered from Pseudomonas putida (S12), but similar approaches may be effective in the characterization of related two-component enzyme systems.

Enantioselective epoxidation of styrene with TBHP catalyzed by bis(oxazoline)–vanadyl–laponite materials

Bis(oxazoline)-vanadyl complexes have been prepared from two precursors, VO(acac)2 and VOSO4, and immobilized on laponite by cation exchange. These catalysts have been tested in the enantioselective epoxidation of styrene with TBHP. The catalytic activities are always much higher in the case of VO(acac)2. The ligand improves the catalytic activity, with similar or slightly better selectivity to styrene oxide (up to 42%) and up to 63% ee in solution. Laponite support is also beneficial in most cases, with selectivities to styrene oxide up to 60%. Moderate enantioselectivities are obtained, better with VOSO4 solids, up to 72% ee.

Enantioselective epoxidation of styrene using in-situ generated dimethyldioxirane and dimeric homochiral Mn(III)-Schiff base complex catalyst

The enantioselective epoxidation of styrene using in-situ generated dimethyldioxirane (DMD) and dimeric homochiral Mn(III)-Schiff base catalyst 1 has been explored. The parameters evaluated were olefin/oxidant molar ration, catalyst loading, reaction temperature and presence of nitrogen coordinating co-catalysts. Conversions of more than 99% and enantiomeric excess 66% were obtained. The selectivity is complete towards styrene oxide using 2 mol% catalyst loading, 1.36 mmol KHSO 5 in 8 ml of water, absence of co-catalyst and 17 °C as reaction temperature. Furthermore, the catalyst could be recycled up to three times with slightly decrease of catalytic activity due mainly to the physical loss of the catalyst during the recovery step and at a lesser extent as a consequence of its oxidative degradation.

N-terminus determines activity and specificity of styrene monooxygenase reductases

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s−1, one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity.

Comparison of Ta–MCM-41 and Ti–MCM-41 as catalysts for the enantioselective epoxidation of styrene with TBHP

Chiral Ti–MCM-41 and Ta–MCM-41 catalysts have been prepared by grafting of Ti(O i Pr) 4 and Ta(OEt) 5 and the modification with R -(+)-diethyl l -tartrate or R -(+)-diisopropyl l -tartrate. In general, the solid catalysts are more active and selective than their homogeneous counterparts in the epoxidation of styrene with tert -butyl hydroperoxide. The enantioselectivities depend on both the nature of the chiral ligand and the calcination temperature of the support, as it is supposed this controls the type of surface species that are formed. The best result of 71% ee is obtained with DIPT–Ta–MCM 550 and is the first example of the use of a Ta catalyst for the enantioselective epoxidation of unfunctionalized alkenes. Nonetheless, the recovered Ta catalysts are less active and selective.

Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequentialternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8s-1. This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

Modified Ti/MCM-41 catalysts for enantioselective epoxidation of styrene

Ti sites grafted on MCM-41, pretreated at different temperatures, have been modified with chiral tartrates, leading to different species. Those solids are active catalyst for the epoxidation of styrene with tert-butyl hydroperoxide, although their efficiency is not optimal, mainly due to the subsequent epoxide rearrangement to phenyl acetaldehyde and further oxidation of it. Styrene oxide is obtained with moderate enantioselectivity, in the range of 55–62% e.e., and the best overall results correspond to the use of MCM-41 calcined at the lowest temperature (550°C). These results are much better with regard to styrene oxide selectivity than those obtained in homogeneous phase under the same conditions, whereas enantioselectivity is similar. The heterogeneous catalysts are recoverable, but only partially due to a loss in activity, although enantioselectivity is kept at similar levels, demonstrating the stability of the supported Ti-tartrate species.

Enantioselective Epoxidation of Styrene by Manganese Chiral Schiff Base Complexes Immobilized on MCM-41.

Two chiral MnIII complexes synthesized and characterized by single-crystal X-ray diffraction were grafted onto mesoporous silica MCM-41. The MCM-41-immobilized solid chiral catalysts were characterized by various spectrochemical and physicochemical methods (XRD, FTIR, 29 Si NMR, TGA, UV/Vis/DRS, EPR, cyclic voltammetry, SEM, BET). Both the homogeneous and heterogeneous MnIII chiral complexes are found to be effective catalysts for epoxidation of styrene. MCM-41-supported chiral catalysts are found to retain their catalytic activity up to five cycles. When an ionic liquid was implemented to enhance the catalytic recyclability of the homogeneous catalysts, they can be recycled up to three times. However, after the third cycle the catalytic activities of the complexes were found to decrease due to rupturing of the chiral catalyst.

Reusable chiral salen Mn(III) complexes with phase transfer capability efficiently catalyze the asymmetric epoxidation of unfunctionalized olefins with NaClO

A series of chiral salen Mn(III) polymers with build-in phase transfer capability was prepared by bridging the chiral salen Mn(III) units with polyethylene glycol (PEG)-based di-imidazolium ionic liquid (IL) side by side. Technologies of characterization suggested the alternation of intact chiral salen Mn(III) unit with PEG-based dicationic imidazolium IL moiety in the rigid one-dimension polymers. Amphipathic nature of PEG-based di-imidazolium IL moiety allowed the obtained catalysts to undergo inherent phase transfer catalysis in asymmetric epoxidation of unfunctionalized olefins with NaClO, which in turn increased the reaction rate of epoxidation in water–dichloromethane biphasic system. Decreasing total length of polyether chain leads to an increase in built-in phase transfer capability of corresponding complex, which further enhances the catalytic performance. 91–97% of conversion was obtained in the enantioselective epoxidation of styrene, α-methylstyrene, indene, 1,2-dihydronaphthalene, 6-cyano-2,2-dimethylchromene, and 6-nitro-2,2-dimethylchromene catalyzed by the complex where number of ethylene oxide unit is 3 within 60min, which is significant higher than that observed for the neat complex (56–74%) and the ICP (62–85%). High enantiomeric excess (ee) for the epoxides (in the range of 67–93%) was also achieved, except for styrene (ee, 34%) and α-methylstyrene (ee, 41%). Furthermore, the efficient phase transfer catalysts could be easily recovered by solvent precipitation and be recycled for seven times without significant loss of the activity and enantioselectivity.

Built-In Regulatory Mechanisms and Active Site Plasticity Open the Door for Engineering the Styrene Catabolic Pathway

Styrene occurs as in the environment as a natural product from the decarboxylation of cinnamic acid and the incomplete combustion of organic material. In recent times increased reliance on the combustion of fossil fuels, and the industrial synthesis of styrene for the production of plastic and rubber products has resulted in dramatic increases in environmental exposure and contamination. Microorganisms evolved from styrene-contaminated environments have developed strategies for detoxification and catabolism of styrene that provide insight for the development of strategies in environmental remediation and biocatalysis.Pseudomonas bacteria employ a styrene-induced three-step enzymatic pathway initiated by the enantioselective epoxidation of styrene to S-styrene oxide by styrene monooxygenase (SMO), a two-component flavoprotein composed of the FAD-specific styrene epoxidase SMOA and NADH-specific flavin reductase, SMOB. Styrene oxide is then isomerized to phenylacetaldehyde by styrene oxide isomerase (SOI), and finally oxidized to phenylacetic acid by an NAD-dependent phenylacetaldehyde dehydrogenase (PADH).In the present work we evaluate the role valine side chains in the catalytic active site of SMOA at positions 211 and 303 in defining the catalytic specificity and efficiency of SMO. Single-turnover stopped-flow fluorescence studies of alanine and isoleucine mutations at these sites indicate that changes in steric bulk primarily impacts the kinetics of the epoxidation reaction with little impact on the subsequent kinetics of elimination of water from the C(4a)-hydroxyflavin intermediate.The styrene metabolic pathway is dependent of the exchange of FAD between the reductase and epoxidase components of SMO and of pyridine nucleotide between the reductase and dehydrogenase. Regulatory reductase-epoxidase and reductase-dehydrogenase interactions will be presented that allow this pathway to be engineered to accommodate a broad range of substrate epoxidation rates while remaining catalytically efficient.

The elpaN-salen series: multifunctional ligands based on d-glucose for the Mn(III)-catalyzed enantioselective epoxidation of styrenes

It is described the simple design of a series of N,N′,O,O′-chiral ligands (elpaN-salen-H2), which represent a subset of the elpaN-type library of ligands based on β-1,2-d-glucodiamine. The corresponding Mn(III) complexes were examined in the asymmetric epoxidation of styrenes with satisfactory results, e.g. cis-β-methylstyrene was functionalised in ees up to 88%.

Asymmetric epoxidation of unfactionalized olefins catalyzed by chiral salen Mn(III) immobilized onto sulfoalkyl modified zirconium poly(styrene-isopropenyl phosphonate)-phosphate and zirconium poly(styrene-phenylvinyl phosphonate)-phosphate

Chiral salen Mn(III) complex has been supported onto sulfoalkyl-modified zirconium poly(styrene-isopropenyl phosphonate)-phosphate and zirconium poly(styrene-phenylvinyl phosphonate)-phosphate (ZPS-IPPA and ZPS-PVPA) by axial coordination. The supported catalysts showed higher chiral induction for enantioselective epoxidation of styrene, α-methylstyrene and indene than its homogeneous chiral salen Mn(III). In addition, ZPS-PVPA-supported catalyst (4c), with a larger pore diameter, was found to be more active than ZPS-IPPA-based catalyst (3c). The influence of the linkage lengths on the catalytic performance was investigated. What's more, the catalysts were easily separated from the reaction systems and could be reused for several times without significant loss of catalytic activity.

Stable chiral salen Mn(III) complexes with built-in phase-transfer capability for the asymmetric epoxidation of unfunctionalized olefins using NaOCl as an oxidant

A series of novel chiral salen Mn(III) complexes with inherent phase-transfer capability were prepared by covalent linkage of the limidazolium IL moieties containing various PEG chains with chiral salen ligand at two sides of 5,5′-position. Technologies of characterization well suggested the presence of polyether chain, the IL linker, and the intact active sites in the complexes. The amphipathic nature of the PEG chain allowed the PEG-based catalysts to undergo built-in phase transfer, which in turn increased the reaction rates in water/organic biphasic systems. Enantioselective epoxidation of styrene, α-methylstyrene, indene, 1,2-dihydronaphthalene, 6-cyano-2,2-dimethylchromene, and 6-nitro-2,2-dimethylchromene catalyzed by the complexes with NaOCl gave >99% conversions within 60min. The enantiomeric excess (ee) for the epoxides was in the range of 68–93%, except for styrene (ee, 35%) and α-methylstyrene (ee, 42%). Furthermore, the PEG-based complexes were stable and could be separated from the reaction mixture by control of the solvent.

Reusable chiral macrocyclic Mn(III) salen complexes for enantioselective epoxidation of nonfunctionalized alkenes

A series of new chiral monomeric and dimeric macrocyclic Mn(III) salen complexes 1–4 with trigol linker were synthesized, characterized (by microanalysis, IR spectroscopy, UV–vis. spectroscopy, optical rotation, and mass spectrometry), and used as catalysts in the enantioselective epoxidation of styrene, cis β-methyl styrene, indene, and chromenes in the presence of several N-oxides as an axial base and NaOCl as an oxidant at 0°C. With the use of chiral dimeric macrocyclic catalyst 3 (2.5mol%), enantio-pure epoxides were achieved in excellent yields (>99%) and enantioselectivities (ee up to 98% in selected cases). The recycling was demonstrated with complex 4 (recyclable up to six cycles studied with retention of enantioselectivity) in the asymmetric epoxidation of styrene. The kinetic investigation with complex 4 for the epoxidation of styrene as the representative substrate showed the first-order dependence on the catalyst and the oxidant but independent on the initial concentration of the substrate.

Rational design of styrene monooxygenase mutants with altered substrate preference

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an α- or β-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of α-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by site-directed mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene.

ChemInform Abstract: Highly Enantioselective Epoxidation of Styrenes Catalyzed by Proline‐Derived C1‐Symmetric Titanium(Salan) Complexes.

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Highly Enantioselective Epoxidation of Styrenes Catalyzed by Proline‐Derived C1‐Symmetric Titanium(Salan) Complexes

Titanium tough! Novel proline-derived C1-symmetric titanium(salan) complexes have been used to catalyze the epoxidation of styrene derivatives with aqueous hydrogen peroxide as the oxidant (see scheme). High enantiomeric excesses ranging from 96 to 98 % were obtained.