High Hydroxylation Activity Research Articles

Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation.

Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) variants can be visually identified via their blue phenotype. Indigo (+) single variants, indigo (-) single variants and a combinatorial library, containing mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. Recombination of indigo (+) single variants to create a multiple-variant library is a particularly useful strategy, as all top performing P450 BM3 variants with high hydroxylation activity were either indigo (+) single variants or contained multiple substitutions. Furthermore, active variants, as determined using the 4-AAP assay, were further characterized and several variants were identified that gave more than 90% conversion with 1,3-dichlorobenzene and predominantly formed 2,6-dichlorophenol; other variants showed significant substrate selectivity. This supports the hypothesis that substitution at positions that enable the indigo (+) phenotype, or hotspot residues, is a general mechanism for increasing aromatic hydroxylation activity. Overall, this research demonstrates that indigo (+) single variants, identified via colorimetric colony-based screening, may be recombined to generate a multiply-substituted variant library containing many variants with high aromatic hydroxylation activity. The combination of colony-based screening and other screening assays greatly accelerates enzyme engineering, as readily-identified indigo (+) single variants can be recombined to create a library of active multiple variants without extensive screening of single variants.

Post-synthesis of MSE-type titanosilicates by interzeolite transformation for selective anisole hydroxylation

MSE-type titanosilicate was efficiently post-synthesized by the combination of interzeolite transformation by siliceous Beta, dealumination and isomorphous substitution of Ti, and it exhibited high catalytic activity in anisole hydroxylation.

Novel bioinspired diiron core complexes with rigid chelating diphosphine ligands for highly efficient catalytic phenol hydroxylation

Hydroquinone (HQ) and catechol (CAT) are the important raw materials in the chemical industry, which can be produced through a direct catalytic hydroxylation process using hydrogen peroxide as an oxidant. Herein, we report two novel diiron core complexes (μ-budt)Fe2(CO)5(DPEphos) (1) and (μ-budt)Fe2(CO)5(Xantphos) (2) supported by rigid heterocyclic xanthene-like diphosphine ligands which exhibit high selectivity and activity in phenol hydroxylation. The structures of complexes 1 and 2 were characterized by FT-IR, UV–vis, 1H NMR, 31P-NMR, X-ray crystallography, while the catalytic products were tested by using HPLC. The FT-IR and UV–vis spectra reveal that two diphosphine ligands exhibit similar impacts on the structure of parent complex (μ-budt)Fe2(CO)6 [budt = SCH(CH3)CH2CH2)S]. Despite the similarity of ligands’ impact on the structure of the parent complex, the catalytic behavior was strongly influenced by the electronic and steric effects. The factors influencing the catalytic activity were optimized, including catalyst dosage, phenol/ H2O2 molar ratio, reaction temperature, and time. Under the optimal conditions, 1 exhibits great catalytic performance with 57.7% phenol conversion and 49.6% DHB yield, both of which are remarkable. The mechanism of phenol hydroxylation using 1 as a catalyst was also proposed. Hydroxyl radicals (•OH) were generated in the catalytic reaction, as demonstrated by electron spin resonance (ESR).

A novel heterogeneous catalyst NH2-MIL-88/PMo10V2 for the photocatalytic activity enhancement of benzene hydroxylation

In this work, heterogeneous catalyst NH2-MIL-88/PMo10V2-3 has shown the high hydroxylation activity of benzene under visible light (a 5 W LED), which mainly attributed to the production of hydroxyl radical(˙OH) and V5+/V4+ redox pair in the existence of electron (e−).

Cation-Exchange Induced Precise Regulation of Single Copper Site Triggers Room-Temperature Oxidation of Benzene.

The controllable synthesis of stable single-metal site catalysts with an expected coordination environment for high catalytic activity and selectivity is still challenging. Here, we propose a cation-exchange strategy for precise production of an edge-rich sulfur (S) and nitrogen (N) dual-decorated single-metal (M) site catalysts (M = Cu, Pt, Pd, etc.) library. Our strategy relies on the anionic frameworks of sulfides and N-rich polymer shell to generate abundant S and N defects during high-temperature annealing, further facilitating the stabilization of exchanged metal species with atomic dispersion and excellent accessibility. This process was traced by in situ transmission electron microscopy, during which no metal aggregates were observed. Both experiments and theoretical results reveal the precisely obtained S, N dual-decorated Cu sites exhibit a high activity and low reaction energy barrier in catalytic hydroxylation of benzene at room temperature. These findings provide a route to controllably produce stable single-metal site catalysts and an engineering approach for regulating the central metal to improve catalytic performance.

Monooxygenation of Nonnative Substrates Catalyzed by Bacterial Cytochrome P450s Facilitated by Decoy Molecules

Cytochrome P450s are a family of heme-containing enzymes that catalyze monooxygenation of inert substrates. Bacterial cytochrome P450s are great candidates as biocatalysts for synthetic applications because of their high hydroxylation activity and their high solubility in water. However, the substrate specificity of bacterial cytochrome P450s is generally high, and their low catalytic activities toward nonnative substrates tends to restrict their application as biocatalysts. We have developed a reaction system that utilizes dummy substrates with structures similar to those of natural substrates, as “decoy molecules”. The decoy molecules induce substrate misrecognition of bacterial P450s, leading to the generation of the active species and ultimately enabling them to catalyze the oxidation of nonnative substrates. The catalytic activity and the enantioselectivity are dependent on the structure of the decoy molecules, suggesting that the reactions can be controlled by variation in the designed structure of ...

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale.

BackgroundHuman mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx).ResultsWe established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L−1 by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library.ConclusionsUnder optimized conditions a maximum productivity of 0.84 g cortisol∗L−1∗d−1 was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0209-5) contains supplementary material, which is available to authorized users.

A Schiff-base-type vanadyl complex grafted on mesoporous carbon nitride: a new efficient catalyst for hydroxylation of benzene to phenol

VO(acac)2 was grafted on mesoporous g-CN material as a Schiff-base-type complex, and showed high catalytic activity in direct hydroxylation of benzene to phenol.

Production of anti-cancer agent using microbial biotransformation.

Microbial biotransformation is a great model system to produce drugs and biologically active compounds. In this study, we elucidated the fermentation and production of an anti-cancer agent from a microbial process for regiospecific hydroxylation of resveratrol. Among the strains examined, a potent strain showed high regiospecific hydroxylation activity to produce piceatannol. In a 5 L (w/v 3 L) jar fermentation, this wild type Streptomyces sp. in the batch system produced 205 mg of piceatannol (i.e., 60% yields) from 342 mg of resveratrol in 20 h. Using the product, an in vitro anti-cancer study was performed against a human cancer cell line (HeLa). It showed that the biotransformed piceatannol possessed a significant anticancer activity. This result demonstrates that a biotransformation screening method might be of therapeutic interest with respect to the identification of anti-cancer drugs.

Cr(III)‐containing Fe3O4/mercaptopropanoic acid‐poly(2‐hydroxyethyl acrylate) nanocomposite: Highly active magnetic catalyst for direct hydroxylation of benzene

ABSTRACTIn this study, polymer‐grafted magnetic nanoparticles containing chromium(III) ions incorporated onto Fe3O4/mercaptopropanoic acid‐poly(2‐hydroxyethyl acrylate) was prepared via a simple and in situ method. The obtained magnetic nanocomposite exhibited high catalytic activity and excellent selectivity in direct hydroxylation of benzene in the presence of hydrogen peroxide under solvent‐free condition. The magnetic catalyst could be also separated by an external magnet and reused seven times without any significant loss of activity/selectivity. Due to the Lewis acidity of the Fe3+ groups in the structure of magnetic nanoparticles, the high efficiency of this catalyst is possibly due to the synergetic effect of Cr3+ and Fe3+ groups in the structure of magnetic nanocomposite. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40383.

Preparation of heterogeneous cationic metalloporphyrin/heteropolyanion composite catalysts and their high catalytic activity in hydroxylation of cyclohexane with molecular oxygen

A new route to improve the metalloporphyrin catalysts is developed, and it is to constitute heterogeneous composite catalysts with immobilized cationic metalloporphyrins and heteropolyanions. By using the method of synchronously synthesizing and immobilizing porphyrins on cross-linked polystyrene microspheres (CPS microspheres), the immobilized porphyrin TAPP-CPS microspheres, on which ternary amine (TA) group-containing phenyl porphyrin (PP) was immobilized, were first prepared, and then the immobilized cationic porphyrin TMPP-CPS microspheres, in whose structure trimethylammoniophenyl porphyrin (TMPP) was contained, were obtained via quaternization reaction. Finally, three immobilized metalloporphyrins, CoTMPP-CPS (shorten as CoP-CPS), MnTMPP-CPS (MnP-CPS) and FeTMPP-CPS (FeP-CPS), were gained through coordination reactions. These immobilized metalloporphyrins were composited with heteropolyanions by right of the mutual electrostatic interaction with phosphotungstic (PW) acid and phosphomolybdic (PMo) acid as reagents, respectively, resulting in several heterogeneous metalloporphyrin/heteropolyanion composite catalysts such as CoPPW-CPS, CoPPMo-CPS and MnPPW-CPS. The composite catalysts were used in the catalytic hydroxylation reaction of cyclohexane with molecular oxygen as oxidant, and their catalytic performances were investigated. The experimental results show that the heterogeneous composite catalysts have extraordinarily high catalytic activity in the hydroxylation reaction of cyclohexane by molecular oxygen, and the cyclohexanol yield in 8 h can get up to 45.5 %. More importantly, the catalytic activity of the heterogeneous composite catalysts is obviously higher than that of the immobilized cationic metalloporphyrins, and the enhanced catalytic activity is originated from a protection of heteropolyanions against the deactivation of metalloporphyrins.

Evidence for Metal–Support Interactions in Au Modified TiOx/SBA-15 Materials Prepared by Photodeposition

Gold nanoparticles have been efficiently photodeposited onto titanate-loaded SBA-15 (Ti(x)/SBA-15) with different titania coordination. Transmission electron microscopy shows that relatively large Au nanoparticles are photodeposited on the outer surface of the Ti(x)/SBA-15 materials and that TiOx tends to form agglomerates in close proximity to the Au nanoparticles, often forming core–shell Au/TiOx structures. This behavior resembles typical processes observed due to strong-metal support interactions. In the presence of gold, the formation of hydrogen on Ti(x)/SBA-15 during the photodeposition process and the performance in the hydroxylation of terephthalic acid is greatly enhanced. The activity of the Au/Ti(x)/SBA-15 materials is found to depend on the TiOx loading, increasing with a larger amount of initially isolated TiO4 tetrahedra. Samples with initially clustered TiOx species show lower photocatalytic activities. When isolated zinc oxide (ZnOx) species are present on Ti(x)/SBA-15, gold nanoparticles are smaller and well dispersed within the pores. Agglomeration of TiOx species and the formation of Au/TiOx structures is negligible. The dispersion of gold and the formation of Au/TiOx in the SBA-15 matrix seem to depend on the mobility of the TiOx species. The mobility is determined by the initial degree of agglomeration of TiOx. Effective hydrogen evolution requires Au/TiOx core–shell composites as in Au/Ti(x)/SBA-15, whereas hydroxylation of terephthalic acid can also be performed with Au/ZnOx/TiOx/SBA-15 materials. However, isolated TiOx species have to be grafted onto the support prior to the zinc oxide species, providing strong evidence for the necessity of Ti–O–Si bridges for high photocatalytic activity in terephthalic acid hydroxylation.

Synthesis of thermal stable mesoporous titanosilicates assembled from titanosilicates precursors at high temperature

A series of mesoporous titanosilicates (MTS-9x) are synthesized in an acidic media from titanosilicate precursors with a polymer surfactant (P123) under temperatures ranging from 100 to 180°C. Characterization and analysis show that the hexagonal mesoporous titanosilicates (MTS-9x) contain both primary and secondary structural building units of TS-1 zeolite in their framework, in a manner similar to MTS-9. Furthermore, 29Si NMR spectral analysis suggests that MTS-9180 synthesized at 180°C promotes a higher condensation of Ti-containing silica networks in the mesoporous walls compared to MTS-9100 synthesized at 100°C. The higher synthesis temperature may also be responsible for the increased thermal stability of the four-coordinated titanium species in MTS-9180, which was verified by UV–vis spectral analysis. Catalytic experiments indicate that MTS-9180 shows high hydroxylation activity, regardless of calcination, for both small organic molecules such as phenol and bulky organic molecules such as 2,3,6-trimethylphenol. Moreover, the recycled catalyst retains a high conversion, confirming the high thermal stability of the titanium species.

Hybrid macromolecular iron and copper complexes in the phenol hydroxylation reaction

Macromolecular iron and copper complexes based on hybrid materials with grafted polyamine polymers have been synthesized. It has been shown that the silica gel-immobilized iron complexes with polyallylamine-supported EDTA and copper complexes with polyethyleneimine exhibit a high activity in phenol hydroxylation with hydrogen peroxide. The catalysts can be recycled without the loss of activity.

Biomimetic Alkene Epoxidation and Alkane Hydroxylation with Sodium Periodate Catalyzed by Mn(III)-salen Supported on Amberlite IRA-200

The Mn(III)-salen, containing phosphonium groups at the 5,5′-positions of the salen ligand supported on Amberlite IRA-200 via electrostatic binding was used for the oxidation of alkenes and alkanes with sodium periodate at room temperature in the presence of imidazoles as axial ligands, and the effect of solvent, different axial ligands, and various oxygen donors was investigated. This heterogenized catalyst shows high catalytic activity in alkene epoxidation and alkane hydroxylation. It showed high selectivity in the epoxidation of stilbenes, α-pinene, and (R)-(+)-limonene, and exhibits a particular ability to epoxidize linear alkenes. The stability and reusability of this new heterogenized metallo-salen complex was also investigated. The catalyst was characterized by FTIR, UV-Vis, SEM, and thermal analysis.

Diatomite as high performance and environmental friendly catalysts for phenol hydroxylation with H2O2

A series of diatomite catalysts were treated and characterized. For the first time, the resulting materials were used in catalysis for the hydroxylation of phenol with H2O2 and showed very high hydroxylation activity due to the Fe species in the diatomite. The effect of HCl treatment, contents of catalysts and H2O2 were investigated and the active components of diatomite were discussed. The results show that diatomite is the promising candidate for industrial output due to their high catalytic activity, easy physical separation and very low costs.

Improvement in thermal stability and catalytic activity of titanium species in mesoporous titanosilicates by addition of ammonium salts

Mesoporous titanosilicates (MTS-9A) with thermally stable titanium species were synthesized by adding ammonium salts in the preparation procedure. Characterization results show that hexagonal mesoporous titanosilicates (MTS-9A) contain primary and secondary structural building units of zeolite in the framework, in a similar manner to MTS-9. The promoted condensation of Ti-containing silica network in MTS-9A leads to higher thermal stability of four-coordinated titanium species compared with MTS-9, as evidenced by UV–vis and 29Si NMR spectra. Catalytic tests indicate that MTS-9A samples show high activity in hydroxylation of both small (phenol) and bulky reactants (2,3,6-trimethylphenol) before and after calcination. Moreover, the recycled catalyst retains high conversion, confirming the high thermal stability of titanium species.

Screening of Compactin-Resistant Microorganisms Capable of Converting Compactin to Pravastatin

A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6beta position of compactin was developed. Agar plates containing different carbon sources and 500 microg compactin mL(-1) were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program.

Phenol hydroxylation using Fe-MCM-41 catalysts

Highly ordered iron-containing mesoporous material, Fe-MCM-41, with 0.5–4 Fe/Si mol% loading was prepared and characterization was performed using XRD, SEM/TEM, EDS, N 2-sorption, and FT-IR and UV–vis spectroscopies. Fe-MCM-41 exhibited high catalytic activity in phenol hydroxylation using H 2O 2 as oxidant, giving phenol conversion of ca. 60% at 50 °C [phenol:H 2O 2 = 1:1, water solvent]. Effects of Fe contents in Fe-MCM-41 and catalyst concentration, temperature, solvent used, phenol/H 2O 2 mole ratios and H 2O 2 feeding method, and catalyst calcination temperature on conversion profiles were examined. Catalyst recycling was performed to investigate the extent of potential metal leaching. Comparisons in performance were also made using nano-sized Fe 2O 3 particles and Fe-salt impregnated MCM-41 as catalyst. Catechol to hydroquinone in product ratio was close to 2:1 in accordance with a free radical reaction scheme involving Fe 2+/Fe 3+ redox pair and the larger amount of Fe species always achieved the given phenol conversion at a shorter reaction time. As the calcination temperature increases from 400 to 800 °C increasing amount of Fe species came out from the MCM-41 framework. Both tetrahedral Fe and extra-framework Fe species were found catalytically active, but high dispersion of Fe species achieved in Fe-MCM-41 was an advantage.

Formation of better catalytically active titanium species in Ti-MCM-41 by vapor-phase silylation

Calcined Ti-MCM-41 was silylated by trimethylchlorosilane in the vapor phase for 2–9 h. 29Si MAS NMR spectroscopic study showed that Ti-MCM-41 silylated for 2–5 h exhibited a peak at 11 ppm, which was assigned to (CH 3) 3SiOSi(OSi) 3 species. Further silylation of Ti-MCM-41 for 5–9 h gave an additional peak at 5.1 ppm, which was assigned to novel titanium species of (CH 3) 3SiOTi(OSi) 3. Ti-MCM-41 silylated for 5 h showed relatively high activity in hydroxylation of benzene and epoxidation of 1-octene compared with calcined Ti-MCM-41. More important, Ti-MCM-41 silylated for 9 h gave much higher activity than Ti-MCM-41 silylated for 5 h. 29Si MAS NMR spectra showed the presence of the titanium species of (CH 3) 3SiOTi(OSi) 3 in Ti-MCM-41 silylated for 5–9 h, and these can be better catalytically active sites than conventional titanium species in Ti-MCM-41.