Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.

HYDROXYLATION OF PHENOL BY IRON(Ⅱ)-PHENANTHROLINE(PHEN)/MCM-41 ZEOLITE

MCM-41 zeolite and Iron (Ⅱ) -Phen/MCM-41 zeolite have been preparedand characterized by XRD,IR, NH3-TPD, BET and UV-Vis. The Iron(Ⅱ)-Phen/MCM-41 zeolite+30% H2O2 system is capable for catalyzing hydroxylation of Phenol.

Hydroxylation Reaction of Phenol to Catechol on Lanthanide-Zirconia Catalyst

The selective H2O2 oxidation reaction of phenol to catechol on Ln-ZrO2 catalyst has improved the selectivity to 85%, to compare with the conventional titania catalyst. With addition of rare earth such as lanthanum, neodymium, to zirconia as catalyst, the selectivity is increased by 72% and 60% respectively in comparison with the bare titania catalyst.

Hydroxylation of Benzene to Phenol via Hydrogen Peroxide in Hydrophilic Triethylammonium Acetate Ionic Liquid

A new Fenton-like system in a medium of hydrophilic triethylammonium type of ionic liquid（IL） was used for the hydroxylation of benzene to phenol. The triethylammonium acetate（[Et3NH][CH3COO]） IL exhibited retardation performance for the decomposition of H2O2 and protection performance for the further oxidation of phenol, thus the yield and selectivity to phenol were promoted greatly. The acidity of the system was proved to be an important factor for the selectivity to phenol. The utilization of H2O2 and the selectivity to phenol, as well as the Turnover number（TON） of the catalyst were effectively enhanced by a benzene-[Et3NH][CH3COO] bi-phase system. The catalyst with [Et3NH][CH3COO] IL was recycled with stable catalytic performance.

Hydroxylation Reaction Mechanism for Nitrosodimethylamine by Oxygen Atom

The hydroxylation reaction mechanism of nitrosodimethylamine（NDMA） by oxygen atom was theoretically investigated at the B3LYP/6-31G＊＊ level. It has been found that the path of the oxydation of the C―H bond is easier than the path involving a Singlet/Triplet crossing. The study of the potential surface shows that both solvent effect at B3LYP/6-31G＊＊ level and different method at more credible MP2/6-311G＊＊ level in the gas phase have no effect on the hydroxylation reaction mechanism. The oxidation hydroxylation process of NDMA by O is exothermic reaction and easy to occur.

Hydroxylation of Phenol Catalyzed by Iron Metal-Organic Framework (Fe-BTC) with Hydrogen Peroxide

Liquid phase catalytic hydroxylation of phenol by Fe-containing metal-organic framework, Fe-BTC (BTC = 1,3,5-benzenetricarboxylate) using 30% H2O2 as an oxidant and H2O as solvent showed good activity and stability under mild reaction conditions. Phenol reacts with hydrogen peroxide over Fe-BTC to produce two main products, viz., catechol and hydroquinone. The effect of temperature, time, substrate/hydrogen peroxide mole ratio and amount of catalyst on catalytic performance were studied. The catalyst could be reused four times without losing significant loss of catalytic performance. The crystallinity and structure of catalyst were unchanged during the catalysis reaction, as confirmed by comparison of XRD and SEM of the fresh and reused catalyst. A reaction mechanism is proposed based on the experimental results.

Preparation of Heteropoly Molybdoselenite Complex and Its Lanthanide Salts and Their Catalysis for Hydroxylation of Phenol

A heteropoly molybdoselenite complex （NH4）6[ Se2Mo8O31 ]·5H2O and its lanthanide salts Ln2 [Se2Mo8O31]-XH2O（Ln = La^3＋, Ce^3＋, Nd^3＋ Sm^3＋） were synthesized and characterized with elemental analysis, IR, UV, XRD and TG-DTA. Their reactivity for hydroxylation of phenol was investigated. The results show that catalytic activity of （ NH4 ）6 [Se2Mo8O31 ]·5H2O is higher than that of its lanthanide salts. The reaction temperatures, the reaction time. the catalvst amount, the ratio of phenol and H2O2 have influence on the phenol conversation, H2O2 selectivity and product distribution. The optimum reaction condition was obtained with orthogonal method. At optimum reaction condition which the ratio of phenol and H2O2 = 1 ： 1, reaction time is 4 h, reaction temperatures is 70℃ and catalyst is 10 g·mol^-1, and the conversation of phenol reaches as high as 29.65 %, the product selectivity can reach 77.2 % for catalyst （NH4）6[ Se2Mo8O31 ]·5H2O.

Hydroxylation of 3-nitrotyrosine by hydroxyl radical

Hydroxylation of 3-nitrotyrosine （3-NT） and 3-NT containing peptide Gly-nitroTyr-Gly in aqueous solution by hydroxyl radical were investigated with gamma irradiation. The structures of the hydroxylated products were confirmed by electrospray ionization mass spectrometry and ^1H NMR spectrometry. The reactivity of 3-nitrotyrosine has been investigated using density functional theory （DFT） calculation.

Effect of Supercritical Fluids on the Biological Activity of Absidia coerulea for the Hydroxylation of Reichsterin's Substance Acetate

The viability and biological activity of Absidia coerulea in compressed or supercritical CO2 and C2H4 were studied. The specific activity of Absidia coerulea in 7.5MPa CO2 and C2H4 at 306K can reach to 23% and 75% respectively, leading to the feasibility of using supercritical C2H4 as an alternative to the organic solvent in the hydroxylation of Reichsterin’s substance acetate.

The Effect of 19-Substituent on the Stereospecific Hydroxylation of △~5-Steroids Using Performic Acid as Oxidant

In the studying of the stereospecific hydroxylation of different Δ5-steroids with performic acid, followed by hydrolysis with CH3OH/KOH, we found 19-substituting groups considerably affected the reaction and we put forward the mechanism.

Electrochemical Probe of Hydroxylation of 4-Nitro-phenol by Cytochrome C with Hydrogen Peroxide

ＥｌｅｃｔｒｏｃｈｅｍｉｃａｌＰｒｏｂｅｏｆＨｙｄｒｏｘｙｌａｔｉｏｎｏｆ４┐Ｎｉｔｒｏ┐ｐｈｅｎｏｌｂｙＣｙｔｏｃｈｒｏｍｅＣｗｉｔｈＨｙｄｒｏｇｅｎＰｅｒｏｘｉｄｅ＊ＺＨＵＹｉ－ｍｉｎ，ＷＡＮＧＪｉｎ－ｃｈｅｎｇ＊＊，ＬＩＪｉｎｇ－ｈｏｎｇａｎ...

PREVOST-WOODWARD HYDROXYLATION OF 3,5-CYCLO-24S-ETHYL-CHOLEST-22-EN-6-ONE

Preost-Woodward hydroxylation of 2 gave the 22S,23S-glycol(3), contrary to expectation.It is suggested that the formation of the two possible iodonium ions(4a and 4b)are reversible,thus allowing the discriminatory acetoxy group to follow the less hindered route,reacting with 4a virtually exclusively.

HYDROXYLATION OF BENZENE BY HETEROPOLY ACIDS(SALTS)CONTAINING VANADIUM

Direct hydroxylation of benzene catalyzed by vanadium-con- taining heteropoly compounds with Keggin or Dawson structure has been investigated.The effects of several factors upon the hydroxylation reaction have also been examined.It is indicated that the highest yield of phenol reached 76%(based on H_2O_(?))with 99.4～100% selectivity.

Microbial transformation for Region-selective Hydroxylation of Z-Butylidenephthalide by Aspergillus niger CGMCC 3.739

Microbial transformation is a useful tool for structure modification of active natural products.Z-Butylidenephthalide is the main active constituent of Angelica sinensis.In the present work,a pair of new oxidized metabolites,namely(-)(11R)-(Z)-11-hydroxy-butylidenephthalide(1a)and(+)(11S)-(Z)-11-hydroxy-butylidenephthalide(1b),were obtained from microbial transformation for region-selective 11-hydroxylation of Z-butylidenephthalide by Aspergillus niger CGMCC 3.739.Their structures,including absolute configurations,were elucidated by extensive analysis of HR-ESI-MS,NMR spectra,and the modified Mosher’s method.This is the first study on the microbial transformation of Z-butylidenephthalide,and these findings offer a tool for region-selective 11-hydroxylation of Z-butylidenephthalide.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

Pyridine-H_5PMo_(10)V_2O_(40) hybrid catalysts for liquid-phase hydroxylation of benzene to phenol with molecular oxygen

Pyridine(Py)-modified Keggin-type vanadium-substituted heteropoly acids(PynPMo10V2O40,n=1 to 5) were prepared by a precipitation method as organic/inorganic hybrid catalysts for direct hydroxylation of benzene to phenol in a pressured batch reactor and their structures were detected by FT-IR.Among various catalysts,Py3PMo10V2O40 exhibits the highest catalytic activity(yield of phenol,11.5%),without observing the formation of catechol,hydroquinone and benzoquinone in the reaction with 80 vol% aqueous acetic acid,molecular oxygen and ascorbic acid used as the solvent,oxidant and reducing reagent,respectively.Influences of reaction temperature,reaction time,oxygen pressure,amount of ascorbic acid and catalyst on yield of phenol were investigated to obtain the optimal reaction conditions for phenol formation.Pyridine can greatly promote the catalytic activity of the Py-free catalyst(H5PMo10V2O40),mostly because the organic π electrons in the hybrid catalyst may extend their conjugation to the inorganic framework of heteropoly acid and dramatically modify the redox properties,at the same time,pyridine adsorbed on heteropoly acids can promote the effect of "pseudo-liquid phase",thus accounting for the enhancement of phenol yield.

Nanotrap-enabled quantification of KRAS-induced peptide hydroxylation in blood for cancer early detection

Circulating peptide is a potential source of biomarkers for cancer detection.However,the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting.Herein,nanotrap fractionation following by mass spectrometry have been applied to quantify the levels of bradykinin (BK) and hydroxylated bradykinin (Hyp-BK) as a relative measure of KRAS-regulated prolyl-4-hydroxylase alpha-1 (P4HA1) which may serve as early diagnosis marker for pancreatic ductal adenocarcinoma (PDAC).We found that P4HA1 can be upregulated by KRASG12v,which is a PDAC driver mutation,using HPNE/KRAS and HPNE cells.And we revealed that P4HA1 is overexpressed in PDAC tumors,compared to normal and inflamed pancreatic tissues.RNA interference revealed that P4HA1 activity was primarily responsible for Hyp-BK production.Mass spectrometry analysis revealed that plasma Hyp-BK/BK ratio was higher in PDAC than pancreatitis patients and healthy controls,while the area under the receiver operating characteristic (ROC) curve (AUC) is 0.8209 (95%Cl,0.7269-0.9149).The Hyp-BK/BK association with PDAC was reproduced in another cohort,where this ratio was found to increase with advancing tumor stage.These novel findings paved the way for wider applications of Nanotrap coupled mass spectrometry as a powerful tool for revealing biosignatures from plasma.

Controlled growth of uniform two-dimensional ZnO overlayers on Au(111)and surface hydroxylation

Ultrathin ZnO nano structures prese nt in teresti ng two-dimensi on al(2D)graphene-like structure in contrast to wurtzite structure in bulk ZnO.Growth on Au(111)has been regarded as a well-established route to the 2D ZnO layers while controlled growth of uniform ZnO nano structures remains as a challe nge.Here,reactive deposition of Zn in O3 and NO2 was employed,which is investigated by sea nning tunneling microscopy and X-ray photoelectr on spectroscopy(XPS).We dem on strate that uniform ZnO mono layer nanoislands and films can be obtained on Au(111)using O3 and uniform ZnO bilayer nanoislands and films form on Au(111)using NO2,respectively.Formation of atomic oxyge n overlayers on Au(111)via exposure to O3 is critical to the formatio n of uniform ZnO mono layer nano structures atop.Near ambient pressure XPS studies revealed that n early full hydroxy lati on occurs on mono layer ZnO structures upon exposure to near ambient pressure water vapor or atomic hydrogen species,while partial surface hydroxylation happens on bilayer ZnO under the same gaseous exposure conditions.

Kinetics and reaction mechanism of phenol hydroxylation catalyzed by La-Cu_4FeAlCO_3

The present work synthesizes La-Cu4FeAlCO3 catalyst under microwave irradiation and characterizes its structure using XRD and IR techniques. The results show that the obtained La-Cu4FeAlCO3 has a hydrotalcite structure. In the phenol hydroxylation with H2O2 catalyzed by La-Cu4FeAlCO3, the effects of reaction time and phenol/H2O2 molar ratio on the phenol hy-droxylation, and relationships between the initial hydroxylation rate with concentration of the catalyst, phenol, H2O2 and reaction temperature are also investigated in details. It is shown the phenol conversion can reach 50.09% (mol percent) in the phenol hydroxylation catalyzed by La-Cu4FeAlCO3, under the reaction conditions of the molar ratio of phenol/H2O2 1/2, the amount ratio of phenol/catalyst 20, reaction temperature 343 K, reaction time 120 min, 10 mL distilled water as solvent. Moreover, a kinetic equation of v = k[La ? Cu 4 FeAlCO 3 ][C 6 H 5 OH][H 2 O 2]. and the activation energy of Ea=58.37 kJ/mol are obtained according to the kinetic studies. Due to the fact that the HO-Cu+-OH species are detected in La-Cu4FeAlCO3/H2O2 system by XPS, the new mechanism about the generation of hydroxyl free radicals in the phenol hydroxylation is pro-posed, which is supposed that HO-Cu+-OH species are transition state in this reaction.

Fabrication and catalytic performance of meso-ZSM-5 zeolite encapsulated ferric oxide nanoparticles for phenol hydroxylation

An encapsulation-structured Fe_(2)O_(3)@mesoZSM-5(Fe@MZ5)was fabricated by confining Fe_(2)O_(3) nanoparticles(ca.4 nm)within the ordered mesopores of hierarchical ZSM-5 zeolite(meso-ZSM-5),with ferric oleate and amphiphilic organosilane as the iron source and meso-porogen,respectively.For comparison,catalysts with Fe_(2)O_(3)(ca.12 nm)encapsulated in intra-crystal holes of meso-ZSM-5 and with MCM-41 or ZSM-5 phase as the shell were also prepared via sequential desilication and recrystallization at different pH values and temperatures.Catalytic phenol hydroxylation performance of the as-prepared catalysts using H_(2)O_(2) as oxidant was compared.Among the encapsulation-structured catalysts,Fe@MZ5 showed the highest phenol conversion and hydroquinone selectivity,which were enhanced by two times compared to the Fe-oxide impregnated ZSM-5(Fe/Z5).Moreover,the Fe-leaching amount of Fe@MZ5 was only 3% of that for Fe/Z5.The influence of reaction parameters,reusability,and ·OH scavenging ability of the catalysts were also investigated.Based on the above results,the structure-performance relationship of these new catalysts was preliminarily described.