Analysis of Monophenol Oxidase Activity Using High Pressure Liquid Chromatography with Electrochemical Detection

Abstract

A very sensitive, specific and reliable quantitative assay was developed for measuring the rate of hydroxylation of tyrosine by mushroom tyrosinase using high pressure liquid chromatography with electrochemical detection (HPLC-ED). The assay employs N-acetyldopamine (NADA) as cofactor and ascorbate as a reducing agent. The product of the reaction, L-dopa (3,4-dihydroxyphenylalanine), was readily separated by HPLC-ED from the remaining interacting components. The reaction was linear with time and proportional to the amount of enzyme present. The amount of ascorbate gradually decreased during the hydroxylation of tyrosine, but as long as ascorbate was present in the reaction mixture the levels of L-dopa and NADA were not altered. The data     

One-Step Hydroxylation of Benzene to Phenol Over Layered Double Hydroxides and their Derived Forms

Phenol, an important bulk organic compound, has diverse applications encompassing both industry and society. Commercially, it is produced through energy intensive three-step cumene process operating at relatively low yield with the co-production of acetone. Several attempts were made for producing phenol through challenging one-step direct hydroxylation of benzene using different oxidants like O₂, N₂O and H₂O₂. Liquid phase hydroxylation of benzene using H₂O₂ found to be more attractive due to its low reaction temperature and environmentally friendly nature (as water is only formed as by-product). The hydroxylation reaction occurs through Fenton’s mechanism; however along with phenol several other products are also formed due to higher reactivity of phenol compared to benzene. Our research group has been working on this reaction for nearly a decade using layered double hydroxides (LDHs) and their derived forms as heterogeneous selective oxidation catalyst. Screening of different LDHs having different metal ions in the layers revealed the necessity of copper for hydroxylation in pyridine. Addition of co-bivalent metal ion along with copper was made in an endeavour to improve the activity that revealed the promising results for CuZnAl LDHs. Efforts were then made to shift from pyridine to environmentally benign solvent, water, for this reaction that showed reasonably good yields with very high selectivity of phenol. Addition of small amount of sulfolane as a co-solvent increased the selectivity for phenol further. The reusability difficulty faced while using as-synthesized LDHs was overcome when calcined LDHs were used. Structure–property-activity relationships were deduced to understand the results observed. The present review besides covering our work also provides the state-of-art on this reaction using different oxidants with emphasis on H₂O₂.     

Formation of Oxygen Radicals in Solutions of Different 7,8‐Dihydropterins: Quantitative Structure‐Activity Relationships

Under certain conditions, 7,8‐dihydroneopterin in aqueous solution promotes hydroxyl‐radical formation. Thus, we investigated the stimulation of hydroxyl‐radical formation by ten different 7,8‐dihydropterins (=2‐amino‐7,8‐dihydropteridin‐4(1H)‐one), i.e., 6‐(1′‐hydroxy) derivatives 1 and 2 , methyl derivatives 3 – 7 , and 6‐(1′‐oxo) derivatives 8 – 10 . All but the 6‐(1′‐oxo) derivatives produced hydroxyl radicals, as measured by the amount of salicylic acid hydroxylation products. This amount was dependent on the stability of the dihydropterin used. In the presence of chelated iron ions, hydroxylation was increased in every case; even 6‐(1′‐oxo) derivatives showed a low hydroxylation of salicylic acid. The degree of increase, however, strongly depended on the side chain of the dihydropterin. The 7,8‐dihydroneopterin ( 2 ) was investigated in more detail. Iron ions influenced both, the stability of 2 and hydroxyl‐radical formation. While iron ions determined the kinetics of the reaction, the amount of 2 was responsible for the amount of hydroxyl radicals formed. Our data establish that promotion of hydroxyl‐radical formation by 7,8‐dihydropterins depends on the oxidizability of the dihydropterins and on their iron‐chelating properties.     

Activation parameters for cyclohexene oxygenation by an oxoiron(IV) porphyrin pi-cation radical complex: entropy control of an allylic hydroxylation reaction

Activation parameters for epoxidation and allylic hydroxylation reactions of cyclohexene with FeIVO(TMP)*+Cl (1) were determined. Within the experimental temperature range, the epoxidation reaction was enthalpy-controlled (i.e., DeltaH > -TDeltaS), while the allylic hydroxylation reaction was entropy-controlled (i.e., -TDeltaS > DeltaH). An unexpectedly large contribution of the entropy term for the allylic hydroxylation reaction indicated that the free energy of activation, DeltaG, rather than the activation energy, Ea, should be used to discuss the reaction mechanism and chemoselectivity. The results of this study bring caution to previous density functional theory studies, in which the reaction mechanism and chemoselectivity are evaluated from calculated Ea.     

气固相反应温度对Ti-ZSM-5物化性能的影响

1983年美国专利US4，410，501报道了钛硅沸石的合成，其独特的催化氧化性能引起了人们极大的关注。但因模板剂四丙基氢氧化铵价格昂贵，而且水热合成条件苛刻，重复性差，限制了其工业应用．因此，人们陆续开发了许多廉价的合成钛硅沸石类催化剂的方法．但是，廉价体系得到的TS-1的催化性能普遍逊色于经典法得到的样品．气固相法是另一种制备钛硅沸石的方法，     

Fe-MSU-1介孔分子筛的合成及其催化苯酚羟基化性能

以月桂醇聚氧乙烯醚为模板剂,在中性条件下成功合成了Fe-MSU-1介孔分子筛.采用X射线衍射、傅里叶变换红外光谱、紫外-可见吸收光谱、电子自旋共振谱、透射电镜、低温N2吸附和电感耦合等离子体原子发射光谱对样品进行了表征.考察了Fe-MSU-1分子筛在苯酚羟基化反应中的催化性能以及工艺条件对苯酚羟基化反应的影响.结果表明,Fe3 离子进入了分子筛骨架,Fe-MSU-1分子筛具有均一的蠕虫状介孔结构,平均孔径约为2.9nm.在焙烧后的Fe-MSU-1样品中,Fe3 主要以四配位的形式存在,无骨架外Fe2O3物种形成.以水为溶剂,在苯酚/H2O2摩尔比为3,反应温度353K,反应时间7h的条件下,苯酚和H2O2的转化率以及苯二酚的选择性分别达到20.4%,96.9%和99.6%.     

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

Phenol hydroxylation is an industrially important reaction, whose main products are catechol and hy-droquinone being diverse applications which are im-portant intermediates for perfumes, drugs, and phar-maceuticals and so on[1]. The processes using H2O2 a…     

钒改性六方介孔硅分子筛催化苯羟基化制苯酚

以偏钒酸铵为钒源,采用溶胶-凝胶法合成了不同钒含量的六方介孔硅（V-HMS）分子筛,利用X射线衍射、N2吸附-脱附和程序升温还原(H2-TPR)对合成的催化剂进行了表征,考察了V-HMS对苯羟基化反应的影响。 结果表明,钒进入了分子筛骨架,并且在HMS分子筛上具有较好的分散性。 V-HMS对苯羟基化反应具有良好的催化活性;高分散的钒氧物种有利于提高苯的羟基化反应性能,溶剂乙腈对反应促进作用明显。 乙腈为溶剂,w(V(5.8)-HMS)=2%,60 ℃反应5 h,苯酚收率达到18.55%,选择性达到100%。     

Iron Incorporated Mesoporous Molecular Sieves Synthesized by a Microwave-Hydrothermal Process and Their Application in Catalytic Oxidation

Fe-FSM-16 and Fe-containing mesoporous materials (Fe-JLU-15) prepared by using semifluorinated surfactant as a template, have been synthesized by microwave-hydrothermal (M-H) process and characterized by several spectroscopic techniques. The catalytic activity of these materials was tested for the phenol hydroxylation and wet phenol oxidation with H₂O₂ under mild reaction conditions. The effect of pH, H₂O₂/PhOH molar ratio and stability of the catalyst on the oxidation process was also investigated. Phenol oxidation and H₂O₂ decomposition show that the Fe-JLU-15 is more active than Fe-FSM-16 and more stable in aqueous solution. The total amount of dissolved iron is less than 5 wt% of the iron initially contained in the catalyst. In phenol hydroxylation, these two solids can effectively catalyze the phenol hydroxylation. Catechol and hydroquinone were observed as the major products, with a difference in the product distribution for these solids. The Fe-JLU-15 has a high selectivity for catechol (63.5 % phenol conversion, CAT/HQ = 2.7) while the Fe-FSM-16 shows a high selectivity for hydroquinone (56.8 % phenol conversion, CAT/HQ < 1) under the same reaction conditions.     

低温晶化温度对B-ZSM-5及Ti-ZSM-5物理化学性质的影响

采用变温晶化方法, 通过改变低温段晶化温度, 水热合成B-ZSM-5沸石. 以B-ZSM-5沸石为母体, 经脱硼处理后, 与TiCl4进行气固相取代反应制得Ti-ZSM-5样品. 采用扫描电子显微镜(SEM)、X射线衍射(XRD)、傅里叶变换红外(FT-IR)光谱、紫外-可见(UV-Vis)光谱、电感耦合等离子体原子发射光谱(ICP-AES)、拉曼光谱、N2吸附-脱附及1,2,4-三甲苯物理吸附等手段对其进行了表征, 并考察了Ti-ZSM-5样品在苯酚羟基化反应中的性能. 结果表明, 合成的母体均为长方体颗粒的聚集体且具有完整的MFI拓扑结构, 但其聚集体颗粒大小、载钛量、孔容及比表面积却存在明显差异; 低温晶化最佳温度为333-353 K, 以此条件下合成出以B-ZSM-5为母体制得的Ti-ZSM-5具有更小的颗粒尺寸、较大的孔容及比表面积, 在苯酚羟基化反应中表现出更加优异的催化性能. 在苯酚与过氧化氢的摩尔比为3的条件下, 苯酚转化率最高可达到20.5%.     

Transition metal substituted polyoxometalates and their application in the direct hydroxylation of benzene to phenol with hydrogen peroxide

A series of transition metal substituted polyoxometalates with a Keggin structure were prepared and utilized for the hydroxylation of benzene to phenol. Among the compounds tested, [(CH₃)₄N]₄PMo₁₁VO₄₀ exhibits the highest phenol yield (13.0%) and selectivity (90.6%) in acetic acid/acetonitrile. Vanadium peroxo is the active site of the reaction, and ammonium also plays an important role. The influence of various reaction parameters, such as solvent, reaction time, reaction temperature, and amount of hydrogen peroxide used were investigated to obtain the optimal reaction conditions.     

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane ( 1 ) to give (S)‐2‐octanol ( 2 ) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C? H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene ( 3 ) to afford (S)‐1‐phenyl‐2‐propanol ( 4 ) with 95 % ee and 98 % subterminal selectivity.     

Aromatic hydroxylation in a copper bis(imine) complex mediated by a micro-eta2:eta2 peroxo dicopper core: a mechanistic scenario

Detailed mechanistic studies on the ligand hydroxylation reaction mediated by a copper bis(imine) complex are presented. Starting from a structural analysis of the CuI complex and the CuII product with a hydroxylated ligand, the optical absorption and vibrational spectra of starting material and product are analyzed. Kinetic analysis of the ligand hydroxylation reaction shows that O2 binding is the rate-limiting step. The reaction proceeds much faster in methanol than in acetonitrile. Moreover, an inverse kinetic isotope effect (KIE) is evidenced for the reaction in acetonitrile, which is attributed to a sterically congested transition state leading to the peroxo adduct. In methanol, however, no KIE is observed. A DFT analysis of the oxygenation reaction mediated by the micro-eta2:eta2 peroxo core demonstrates that the major barrier after O2 binding corresponds to electrophilic attack on the arene ring. The relevant orbital interaction occurs between the sigma* orbital of the Cu2O2 unit and the HOMO of the ligand. On the basis of the activation energy for the rate-limiting step (18.3 kcal mol(-1)) this reaction is thermally allowed, in agreement with the experimental observation. The calculations also predict the presence of a stable dienone intermediate which, however, escaped experimental detection so far. Reasons for these findings are considered. The implications of the results for the mechanism of tyrosinase are discussed.     

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L⁻¹ in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.     

Steric factors override thermodynamic driving force in regioselectivity of proline hydroxylation by prolyl-4-hydroxylase enzymes

Prolyl-4-hydroxylase is an important nonheme iron-containing dioxygenase in humans involved in the regioselective hydroxylation of a proline residue in a peptide chain on the C(4) position. In biosystems this process is important to create collagen cross-linking and cellular responses to hypoxia. We have performed a series of density functional theory (DFT) studies into the origin of the regioselectivity of proline hydroxylation by P4H enzymes using a minimal active site model (where substrate is unhindered in the binding site) and a larger active site model that incorporates steric hindrance of the substrate by several secondary sphere aromatic residues. Our studies show that thermodynamically the most favorable hydrogen atom abstraction position of proline is from the C(5) position; hence, the small model gives a low reaction barrier and large exothermicity for this process. However, stereochemical repulsions of the substrate with aromatic residues of Tyr(140) and Trp(243) in the second coordination sphere prevent C(5) hydroxylation and make C(4) hydroxylation the dominant mechanism, despite a lesser driving force for the reaction. These studies explain the remarkable regioselectivity of proline hydroxylation by P4H enzymes and show that the regioselectivity is kinetically controlled but not thermodynamically. In addition, we calculated spectroscopic parameters and found good agreement with experimental data.     

Copper-mediated regio- and stereoselective 12β-hydroxylation of steroids with molecular oxygen and an unexpected 12β-chlorination

It is shown, that copper(I) complexes of 17-(2-iminomethyl)pyridino steroids (17-IMPY steroids) can react with molecular oxygen followed by a regio- and stereoselective γ-hydroxylation in 12β-position. After decomplexation and hydrolysis of the IMPY group 12β-hydroxy-17-ketones are available in practical useful yields. IMPY compounds are simple to prepare by condensation of oxo compounds with (2-aminomethyl)pyridine. In the cases of 17-IMPY steroids the yields in the hydroxylation procedure of an unactivated CH₂ group are higher by starting with copper(II) complexes, reduction with benzoin/triethylamine in acetone and reaction with molecular oxygen in comparison to the direct reaction of copper(I) complexes with molecular oxygen in acetone. Employing the procedure in dichloromethane as solvent starting with copper(II) complexes surprisingly the 12β-chloro compound could be isolated next to the hydroxylation product. This regio- and stereoselective γ-chlorination takes place also in acetone, when triethylammonium chloride is added to the reaction mixture. Oxygen is necessary for this reaction. The mechanistic and stereochemical aspects of these new reactions are discussed. Comparison of the different yields of steroids with different A-ring [3-methoxy-estra-1,3,5(10)-triene and 3β-hydroxy-androst-5-ene] pointed out to a subtle influence of the molecular structure far from the reaction centre on these reactions. The successful hydroxylation of the IMPY derivative of 3β-hydroxy-androst-5-ene-17-one shows the tolerance of a homoallylic system against this oxidation procedure. By Oppenauer oxidation 12β-hydroxy-androst-4-ene-3,17-dione is available. The hydroxylation procedure opens a short way to 12β-hydroxy-17-oxo steroids, which are difficult to obtain by other routes.     

Kinetics and reaction mechanism of phenol hydroxylation catalyzed by La-Cu<Subscript>4</Subscript>FeAlCO<Subscript>3</Subscript>

The present work synthesizes La-Cu₄FeAICO₃ catalyst under microwave irradiation and characterizes its structure using XRD and IR techniques. The results show that the obtained La-Cu₄FeAICO₃ has a hydrotalcite structure. In the phenol hydroxylation with H₂O₂ catalyzed by La-Cu₄FeAICO₃, the effects of reaction time and phenol/H₂O₂ molar ratio on the phenol hydroxylation, 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-Cu₄FeAICO₃, under the reaction conditions of the molar ratio of phenol/H₂O₂1/2, 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-Cu₄FeAlCO₃][C₆H₅OH][H₂O₂]. and the activation energy of E _a=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-Cu₄FeAICO₃/H₂O₂ system by XPS, the new mechanism about the generation of hydroxyl free radicals in the phenol hydroxylation is proposed, which is supposed that HO-Cu⁺-OH species are transition state in this reaction.     

中孔氧化硅负载铬催化剂上苯高选择性生成苯酚：响应曲面分析法用于反应条件优化

以硝酸铬为前驱体,中孔氧化硅SBA-16为载体,采用简单浸渍法制备了Cr/SBA-16催化剂,并采用广角和小角X射线衍射、N2吸附-脱附、透射电镜和紫外-可见光谱等技术对其进行了表征.同时将该催化剂用于以H2O2为氧化剂的苯直接羟基化制苯酚反应以考察其催化性能.将中心组合设计与响应曲面分析法(RSM)相结合,对影响反应性能的操作变量如反应温度、反应时间及H2O2和催化剂用量进行了优化.结果表明,独立变量和苯酚产率之间的关系可用二阶多项式模型来表达,其相关系数(R2)高达0.985,表明用RSM预测的数值与实验值吻合较好.得到的苯酚选择性较高时的操作条件为：反应温度324 K,反应时间8 h, H2O2和催化剂用量分别为3.28 mL和0.09 g.由此可见,将RSM法用于苯羟基化制苯酚反应条件优化是可靠的.     

细胞色素P450催化4-氯-N-环丙基-N-异丙基苯胺Ca-H羟基化反应机理的理论研究

采用密度泛函理论的B3LYP方法,研究了细胞色素P450催化4-氯-N-环丙基-N-异丙基苯胺Ca-H羟基化的反应机理,该反应包含环丙基的羟基化和异丙基的羟基化两个反应途径,且这两个反应路径都是包含氢原子传递的协同过程,二重态的能垒明显低于四重态,反应主要在二重态上进行.通过比较这两个反应路径中Ca-H羟基化反应的活化...     

Efficient hydroxylation of aromatic compounds catalyzed by an iron(II) complex with H2O2

A mononuclear iron(II) complex, Et₄N[Fe(C₁₀H₆NO₂)₃], coordinated by three 1‐nitroso‐2‐naphtholate ligands in a fac‐N₃O₃ geometry, was initiated to catalyze the direct hydroxylation of aromatic compounds to phenols in the presence of H₂O₂ under mild conditions. Various reaction parameters, including the catalyst dosage, temperature, mole ratio of H₂O₂ to benzene, reaction time and solvents which could affect the hydroxylation activity of the catalyst, were investigated systematically for benzene hydroxylation to obtain ideal benzene conversion and high phenol distribution. Under the optimum conditions, the benzene conversion was 10.2% and only phenol was detected. The catalyst was also found to be active towards hydroxylation of other aromatic compounds with high substrate conversions. The hydroxyl radical formed due to the reaction of the catalyst and H₂O₂ was determined to be the crucial active intermediate in the hydroxylation. A rational pathway for the formation of the hydroxyl radical was proposed and justified by the density functional theory calculations. Copyright © 2014 John Wiley & Sons, Ltd.