The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)⁻¹.min⁻¹) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.     

Engineering P450 Peroxygenase to Catalyze Highly Enantioselective Epoxidation of cis‐β‐Methylstyrenes

P450 119 peroxygenase and its site‐directed mutants are discovered to catalyze the enantioselective epoxidation of methyl‐substituted styrenes. Two new site‐directed P450 119 mutants, namely T213Y and T213M, which were designed to improve the enantioselectivity and activity for the epoxidation of styrene and its methyl substituted derivatives, were studied. The T213M mutant is found to be the first engineered P450 peroxygenase that shows highly enantioselective epoxidation of cis‐β‐methylstyrenes, with up to 91 % ee. Molecular modeling studies provide insights into the different catalytic activity of the T213M mutant and the T213Y mutant in the epoxidation of cis‐β‐methylstyrene. The results of the calculations also contribute to a better understanding of the substrate specificity and configuration control for the regio‐ and stereoselective peroxygenation catalyzed by the T213M mutant.     

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C−H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H₂O₂ complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H₂O₂ complex which would be more abundant in the peroxide-driven reactions.     

Non-covalent modification of the active site of cytochrome P450 for inverting the stereoselectivity of monooxygenation

The enantioselectivity in the sulfoxidation of thioanisole catalyzed by cytochrome P450_BSβ with a decoy molecule, a dummy molecule of the natural substrate, can be inverted by changing the structure of the decoy molecule. The methodology demonstrated herein shows the potential for controlling the stereoselectivity of biocatalysts without any mutagenesis.     

Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min⁻¹ P450BM3⁻¹ and 40 200 P450BM3⁻¹ when N‐heptyl‐l ‐proline modified with l ‐phenylalanine (C7‐l ‐Pro‐l ‐Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild‐type P450BM3. This method for non‐native substrate hydroxylation by wild‐type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.     

Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (k _cat/K _m) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3-hydroxy-indole) compared to parent mutant (93 vs72%).     

Stabilizing DNAzymes through Encapsulation in a Metal–Organic Framework

DNAzymes are a promising class of bioinspired catalyst; however, their structural instability limits their potential. Herein, a method to stabilize DNAzymes by encapsulating them in a metal–organic framework (MOF) host is reported. This biomimetic mineralization process makes DNAzymes active under a wider range of conditions. The concept is demonstrated by encapsulating hemin-G-quadruplex (Hemin-G4) into zeolitic imidazolate framework-90 (ZIF-90), which indeed increases the DNAzyme's structural stability. The stabilized DNAzymes show activities in the presence of Exonuclease I, organic solvents, or high temperature. Owing to its elevated stability and heterogeneous nature, it is possible to perform catalysis under continuous-flow conditions, and the DNAzyme can be reactivated in situ by introducing K⁺. Moreover, it is found that the encapsulated DNAzyme maintains its high enantiomer selectivity, demonstrated by the sulfoxidation of thioanisole to (S)-methyl phenyl sulfoxide. This concept of stabilizing DNAzymes expands their potential application in chemical industry.     

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

We report a unique strategy for the development of a H₂O₂‐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 H₂O₂. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H₂O₂ system previously reported. This work provides the first example of the activation of the normally H₂O₂‐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.     

Highly Efficient Biomimetic Oxidation of Sulfide to Sulfone by Hydrogen Peroxide in the Presence of Manganese meso‐Tetraphenylporphyrin

Low amount of manganese meso‐tetraphenyl porphyrin [Mn(TPP)] was used for highly efficient selective oxidation of sulfide to sulfone by hydrogen peroxide at room temperature. Sulfones were produced directly with yields generally around 90% while the catalyst concentration was only 4×10^?5 mol·L^?1. In a large‐scale experiment of thioanisole oxidation, the isolated yield of sulfone (87%) was obtained and the turnover number (TON) reached up to 8×10⁶, which is the highest TON for the oxidation systems of sulfide to sulfone catalyzed by metalloporphyrins.     

Hemoglobin,horseradish peroxidase,and heme-bovine serum albumin as biocatalyst for the oxidation of dibenzothiophene

Hemoglobin, horseradish peroxidase, and bovine serum albumin incubated heme-catalyzed the oxidation of dibenzothiophene into sulfoxide in the presence of hydrogen peroxide. This reaction was carried out in an aqueous buffer containing 25% of water-miscible organic solvents. The observation of this transient state of hemoproteins during sulfoxidation showed heme degradation. None of the compounds usually involved in a classical peroxidative activity mechanism were detected. Furthermore, this activity did not appear to be based on a Fenton-type reaction. The highest degrees of sulfoxidation were obtained with hemoglobin. Under the best conditions of reaction, 100% of dibenzothiophene were converted into dibenzothiophene sulfoxide by hemoglobin. Heat-denatured hemoproteins did keep their sulfoxidation activity. With hemoglobin, a k_cat of 0.22 min^-1 was determined. Nearly the same values were obtained with heat-denatured hemoglobin and bovine serum albumin-adsorbed heme. With horseradish peroxidase, only 4% of conversion was attained. This percentage could be slightly increased by using a less pure peroxidase or heat-denatured peroxidase.     

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

A series of 18‐fluoro thiastearates were prepared and incubated with a yeast Δ9‐desaturating system. The relative efficiency of desaturase‐mediated sulfoxidation was monitored via ¹⁹F‐NMR analysis of the sulfoxide products, and a strong preference for oxo transfer to the S‐atom occupying the 9‐position was confirmed. The oxidation profile obtained in this manner matched that of analogous experiments with non‐fluorinated substrates. These results form the basis of a versatile ¹⁹F‐NMR‐based method for mapping the position of the putative diiron oxidant relative to substrate, and has potential application to the study of membrane‐bound desaturases in vitro.     

Oxidation of acyclic monoterpenes by P450 BM-3 monooxygenase: influence of the substrate E/Z-isomerism on enzyme chemo- and regioselectivity

Oxidized terpenes and terpenoids are highly valuable compounds for organic chemistry. Cytochrome P450 monooxygenase P450 BM-3 from Bacillus megaterium is able to catalyze oxidation of terpenes with high efficiency. Mutations at the amino acid positions 47, 51, and 87 resulted in significantly enhanced activity and regioselectivity of the enzyme during oxidation of geranylacetone and related compounds. The activity of the mutant R47L/Y51F/F87V was in the order of ketone>alcohol>aldehyde>acid. An effect of the substrate cis/trans-isomerism on the enzyme chemo- and regioselectivity was studied. P450 monooxygenase demonstrated similar NADPH turnovers with cis/trans isomers, nerylacetone/geranylacetone (1.9×10³/2.1×10³ min⁻¹) and nerol/geraniol (5.7×10²/5.9×10² min⁻¹), however, resulted in different number of products and product distribution. The Z-isomers, nerylacetone and nerol, were oxidized resulting in several products (five and three, respectively), including allylic alcohols. In contrast, E-isomers were epoxidized exclusively. Geranylacetone was converted with high activity (2080 min⁻¹) and enantioselectivity (97% ee) to 9,10-epoxygeranylacetone, while geraniol was enantioselectively epoxidized to the 6,7-epoxide (250 min⁻¹, 90% ee) with 90% regioselectivity.     

Water-soluble polymer anchored peroxotitanates as environmentally clean and recyclable catalysts for mild and selective oxidation of sulfides with H2O2 in water

Anchoring of peroxotitanium (pTi) species to linear water-soluble acrylic acid based polymers, poly(sodium acrylate) (PA) and poly(sodium methacrylate) (PMA) led to the successful synthesis of a pair of new, water-tolerant and recyclable catalysts of the type [Ti₂(O₂)₂O₂(OH)₂]^4-—L (L = PA or PMA), highly effective in chemoselective sulfoxidation of organic sulfides with 30% H₂O₂ in aqueous medium at ambient temperature. The catalytic protocol is high yielding (TOF up to 11,280 h^?1), operationally simple as well as environmentally clean and safe, being free from halide, or any other toxic auxiliaries. The catalysts are sufficiently stable to afford easy recyclability for at least 10 consecutive reaction cycles of sulfoxidation with consistent activity selectivity profile. Oxidation of dibenzothiophene (DBT) to respective high purity sulfoxide or sulfone could also be accomplished using the same catalysts by variation of reaction conditions.     

2,10－莰烷二醇衍生的钛络合物催化的不对称硫醚氧化及其机理

在2,10－莰烷二醇/钛催化的亚砜化反应中,枯烯过氧化氢(CHP)和叔丁基过氧化氢(TBHP)分别给出R构型和S构型亚砜。在动力学拆分过程中,用CHP作氧化剂导致亚砜的构型发生逆转,但是用TBHP则保留不变。基于这些结果和电喷雾质谱(ESI－MS)数据,亚砜化反应的机理推测为分子内亲核氧转移到络合的硫醚底物上。     

P450Jα: A New,Robust and α-Selective Fatty Acid Hydroxylase Displaying Unexpected 1-Alkene Formation

Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450_Jα) which catalyses the selective conversion of a broad range of FAs (C6:0-C16:0) and oleic acid (C18:1) with H₂O₂ as oxidant. Under optimized reaction conditions P450_Jα yields α-hydroxy acids all with >95 % regioselectivity, high specific activity (up to 15.2 U mg⁻¹) and efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respect to productivity (TON=394 min⁻¹). On preparative scale, conversion of C12:0 reached 83 % (0.9 g L⁻¹) when supplementing H₂O₂ in fed-batch mode. Under similar conditions P450_Jα allowed further the first biocatalytic α-hydroxylation of oleic acid (88 % conversion on 100 mL scale) at high selectivity and in good yields (1.1 g L⁻¹; 79 % isolated yield). Unexpectedly, P450_Jα displayed also 1-alkene formation from shorter chain FAs (≤C10:0) showing that oxidative decarboxylation is more widely distributed across this enzyme family than reported previously.     

Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.     

Enantioselective Sulfoxidations Catalyzedby Horseradish Peroxidase, ManganesePeroxidase, and Myeloperoxidase

 Horseradish peroxidase (HRP), myeloperoxidase (MPO), and manganese peroxidase (MnP) have been shown to catalyze the asymmetric sulfoxidation of thioanisole. When H₂O₂ was added stepwise to MPO, a maximal yield of 78% was obtained at pH 5 (ee 23%), whereas an optimum in the enantiomeric excess (32%, (R)-sulfoxide) was found at pH 6 (60% yield). For MnP a yield of 18% and a high enantiomeric excess of 91% of the (S)-sulfoxide were obtained at pH 5 and a yield of 36% and an ee of 87% at pH 7.0. Optimization of the conversion catalyzed by horseradish peroxidase at pH 7.0 by controlled continuous addition of hydrogen peroxide during turnover and monitoring the presence of native enzyme as well as of intermediates I, II, and III led to the formation of the sulfoxide in high yield (100%) and moderate enantioselectivity (60%, (S)-sulfoxide).     

NHC copper complexes functionalized with sulfoxide and sulfone moieties

A series of N‐heterocyclic copper carbene complexes bearing sulfoxide and sulfone moieties have been prepared. In case of new copper compounds with sulfone ligand, the solid‐state structures were determined using X‐Ray crystallography. Obtained complexes were investigated as catalysts in such transformations as: 1,3‐dipolar cycloaddition of alkynes and azides (CuAAC), A³ coupling reaction and β‐hydroboration and compared with standard copper catalytic systems.     

Enantioselective Sulfoxidations Catalyzedby Horseradish Peroxidase, ManganesePeroxidase, and Myeloperoxidase

Summary.  Horseradish peroxidase (HRP), myeloperoxidase (MPO), and manganese peroxidase (MnP) have been shown to catalyze the asymmetric sulfoxidation of thioanisole. When H₂O₂ was added stepwise to MPO, a maximal yield of 78% was obtained at pH 5 (ee 23%), whereas an optimum in the enantiomeric excess (32%, (R)-sulfoxide) was found at pH 6 (60% yield). For MnP a yield of 18% and a high enantiomeric excess of 91% of the (S)-sulfoxide were obtained at pH 5 and a yield of 36% and an ee of 87% at pH 7.0. Optimization of the conversion catalyzed by horseradish peroxidase at pH 7.0 by controlled continuous addition of hydrogen peroxide during turnover and monitoring the presence of native enzyme as well as of intermediates I, II, and III led to the formation of the sulfoxide in high yield (100%) and moderate enantioselectivity (60%, (S)-sulfoxide). Received November 18, 1999. Accepted January 21, 2000     

Dérivés Alléniques de l'Étain,du Plomb et du Mercure. Signes Relatifs des Constantes de Couplage Métal-Proton á Travers Deux et Quatre Liaisons

Satellites corresponding to metal-proton coupling constants through two and four bonds are observed in PMR spectra of Pb, Sn and Hg allenic derivatives. The relative signs of these coupling constants are deduced from analysis of the satellite spectra: ²J(X? H) and ⁴J(X? H) are of opposite signs for X = ²⁰⁷Pb, ¹¹⁹Sn, ¹¹⁷Sn and of same sign for X = ¹⁹⁹Hg. Probable absolute signs of reduced coupling constants are discussed in relation to published data: ²K(X? C? H) is probably positive for X = ²⁰⁷Pb, ¹¹⁹Sn, ¹¹⁷Sn and ¹⁹⁹Hg. ⁴K(X? C?C?C? H) is probably negative for X = ²⁰⁷Pb, ¹¹⁹Sn, ¹¹⁷Sn and positive for X = ¹⁹⁹Hg.