Kinetics of epoxidation by a Musa paradisiaca chloroperoxidase

Epoxidations of indene, styrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 4-bromostyrene, and naphthalene using Musa paradisiaca plant juice chloroperoxidase in the presence of H₂O₂ and t-butyl peroxide as oxidants have been studied. The steady-state kinetic parameters, K_m and k_cat of the enzyme for the above substrates have been determined. The temperature and pH optima of the epoxidation are 25°C and 6.2, respectively. The yield of styrene oxide in the presence of H₂O₂ was 44%. The results show that M. paradisiaca plant juice chloroperoxidase is a potential biocatalyst for organic epoxidation reactions.     

4-Chlorophenol degradation by chloroperoxidase from Caldariomyces fumago

This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl⁻ containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5–50 and 0.005–50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H₂O₂ (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.     

Asymmetric oxidation of 1,3-cyclohexadiene catalysed by chloroperoxidase from Caldariomyces fumago

Chloroperoxidase from Caldariomyces fumago catalyses the oxidation of prochiral 1,3-cyclohexadiene using TBHP as terminal oxidant. The process occurs enantioselectively and furnishes the non-racemic trans diols 1,2- and 1,4-dihydroxycyclohexene, (−)-3 and (+)-4, in good ee and yield.     

Isomeric oxabenzochrysenones from Musa acuminata and Wachendorfia thyrsiflora

Two new oxabenzochrysenones (naphthoxanthenones), representing phenylphenalenone-related natural products, were isolated from Musa acuminata and Wachendorfia thyrsiflora.     

51V NMR chemical shifts calculated from QM/MM models of vanadium chloroperoxidase

(51)V NMR chemical shifts calculated from QM/MM-optimized (QM=quantum mechanical; MM=molecular mechanical) models of vanadium-dependent chloroperoxidase (VCPO) are presented. An extensive number of protonation states for the vanadium cofactor (active site of the protein) and a number of probable positional isomers for each of the protonation states are considered. The size of the QM region is increased incrementally to observe the convergence behavior of the (51)V NMR chemical shifts. A total of 40 models are assessed by comparison to experimental solid-state (51)V NMR results recently reported in the literature. Isotropic chemical shifts are found to be a poor indicator of the protonation state; however, anisotropic chemical shifts and the nuclear quadrupole tensors appear to be sensitive to changes in the proton environment of the vanadium nuclei. This detailed investigation of the (51)V NMR chemical shifts computed from QM/MM models provides further evidence that the ground state is either a triply protonated (one axial water and one equatorial hydroxyl group) or a doubly protonated vanadate moiety in VCPO. Particular attention is given to the electrostatic and geometric effects of the protein environment. This is the first study to compute anisotropic NMR chemical shifts from QM/MM models of an active metalloprotein for direct comparison with solid-state MAS NMR data. This theoretical approach enhances the potential use of experimental solid-state NMR spectroscopy for the structural determination of metalloproteins.     

手性环氧氯丙烷的CPO酶催化不对称合成

基于氯过氧化物酶(CPO)对有机底物的手性识别,以CPO催化、叔丁基过氧化氢(TBHP)氧化3-氯丙烯合成手性(R)-环氧氯丙烷,并引入多羟基化合物为添加剂提高了目标产物的产率及对映选择性.反应主要受体系的pH值以及CPO用量等因素控制.UV-vis及CD光谱分析表明,反应体系中引入多羟基化合物(甘油、PEG400、PEG600)时,CPO的血红素辅基暴露程度增加,底物容易接近活性中心,同时CPO的α-螺旋结构得以加强,从而有效提高了产物收率.CPO对底物的手性识别主要基于底物与酶催化中间体([Fe(IV)=O·]+)形成的复合物对酶的稳定性的影响.通过反应条件优化,(R)-环氧氯丙烷产率可达67.3%,对映选择性(ee)97.5%.     

Purification and structural determination of hematopoietic stem cell-stimulating monoacetyldiglycerides from Cervus nippon (deer antler)

A mixture of monoacetyldiglycerides was newly isolated from the chloroform extract of antlers of Cervus nippon, guided by the hematopoietic stimulation of stem cells. The structures of monoacetyldiglycerides were determined by various spectroscopic methods: FAB MS, CID tandem MS, and 1D and 2D NMR. A mixture of at least nine inseparable sn-3-monoacetyldiglycerides was identified: 1 [C(39)H(72)O(6) (C16 : 0/C18 : 1)], 2 [C(39)H(72)O(6) (C18 : 1/C16 : 0)], 3 [C(39)H(70)O(6) (C16 : 0/C18 : 2)], 4 [C(39)H(70)O(6) (C18 : 2/C16 : 0)], 5 [C(41)H(74)O(6) (C18 : 0/C18 : 2), 6 [C(41)H(74)O(6) (C18 : 2/C18 : 0)], 7 [C(41)H(74)O(6) (C18 : 1/C18 : 1)], 8 [C(43)H(74)O(6) (C18 : 0/C20 : 4)], and 9 [C(43)H(74)O(6) (C20 : 4/C18 : 0)]. Among these nine monoacetyldiglycerides in deer antlers, compound 3 was one of the major compounds and was efficiently synthesized from glycerol. Spectral data of synthetic monoacetyldiglyceride 3 were compared with the corresponding data for the mixture of natural monoacetyldiglycerides. The mixture of natural monoacetyldiglycerides from deer antlers showed potent activity on the hematopoiesis (stimulation index=1.40+/-0.05, p<0.02 at 1 microg/ml), and synthetic monoacetyldiglyceride 3 showed even better activity (stimulation index=1.54+/-0.12, p<0.001, at 1 microg/ml).     

Identification of intermediates in the catalytic cycle of chloroperoxidase

Background: Chloroperoxidase (CPO) is the most versatile of the hemethiolate proteins, catalyzing the chlorination of activated CH bonds and reactions reminiscent of peroxidase, catalase, and cytochrome P450. Despite 30 years of continuous efforts, no intermediates of the enzyme's catalytic cycle have been identified except for compound I. Thus, in the absence of conclusive evidence it is generally believed that the halogenation of substrates proceeds by means of ‘free HOCl’ in solution.Results: The pH profile of chloroperoxidase from Caldariomyces fumago revealed a new active-site complex that can be detected only at pH 4.4. According to ultra-violet (UV) spectroscopy, and by comparison with suitable enzyme models, this intermediate is the HOCl adduct of the iron(III) protoporphyrin(IX). Inactivation of chloroperoxidase by diethyl pyrocarbonate, which interrupts the proton shuttle by modification of the distal histidine, led to the formation of the ⁻OCl adduct of the iron complex, which was identified by comparison with a corresponding active site analogue.Conclusions: The availability of enzyme models of heme-thiolate proteins allowed the identification by UV spectroscopy of both the ⁻OCl adduct and the HOCI adduct of the iron(III) protoporphyrin(IX) of chloroperoxidase. The existence of these previously elusive intermediates suggests that the chlorination catalyzed by CPO, and its corresponding active site analogue, proceeds by Cl⁺ transfer from the HOCl adduct to the substrate bound in the distal pocket of the enzyme.     

Novel applications of chloroperoxidase: enantioselective oxidation of racemic epoxyalcohols

The CPO-catalysed enantioselective oxidations of racemic glycidol and cis-2,3-epoxyhexanol to the corresponding aldehydes were studied using tert-butyl hydroperoxide in the 9:9:1 and 18:1 mixtures of hexane:ethyl acetate:buffer and hexane:buffer, respectively. Temperature and pH (100 mM citrate buffer, pH 4.0–7.0) effects on enantioselectivity in the terms of ee for the less reactive alcohol enantiomers with conversion were studied.     

Electrochemical studies of chloroperoxidase on poly-L-lysine film modified GC electrode

<正>Poly-L-lysine(PLL) was first electrodeposited onto the surface of a glassy carbon(GC) electrode.The PLL modified electrode was used to immobilize chloroperoxidase(CPO) via 1-[(3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(EDC).The electrochemical behaviors of immobilized CPO on PLL/GC electrode were investigated by cyclic voltammetry(CV).The CV results obtained showed that CPO was successfully immobilized on the PLL/GC electrode and a fast direct electron transfer between CPO and PLL-GC electrode was achieved with a formal redox potential of -0.23 V vs.SCE.The CPO-PLL/GC modified electrode showed a good catalytic activity for electrocatalytical reduction of O_2,promising for a broad range of CPO-catalyzed transformations.     

Phenolic constituents from the fruit juice of Flacourtia inermis

A chemical investigation of the fruit juice of Flacourtia inermis furnished five caffeoylquinic acid derivatives: methyl chlorogenate (1), methyl 5-O-caffeoylquinate (2), methyl 4-O-caffeoylquinate (3), n-butyl chlorogenate (4), n-butyl 5-O-caffeoylquinate (5) and a rare phenolic glucoside (rel)-6α-benzoyloxy-1α,2α-dihydroxy-5-oxocyclohex-3-enecarboxylic acid 2-(6-O-benzoyl-β-D-glucopyranosyloxy)-5-hydroxybenzyl ester (6), together with quinic acid (7) and malic acid (8). Compounds 1, 2, 4 and 5 showed strong radical scavenging properties towards the 2,2'-diphenyl-1-picrylhydrazyl radical.     

Ionic liquid modified GC electrode for the direct electrochemistry of chloroperoxidase

Chloropcroxidase （CPO） was immobilized by konjac glucomannan （KGM） on the 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4]/Nafion modified glassy carbon eloctrode. The electrochemical behaviors of the immobilized CPO were investigated by cyclic voltammetry. The results showed that CPO was successfully immobilized on the GCE and underwent fast direct electron transfer reactions with the formal potential at -0.3 V vs. SCE. The modified electrode showed a good catalytic activity for elcctrocatalytical reduction of O2 and H2O2.     

Application of hydrophilic ionic liquids as co-solvents in chloroperoxidase catalyzed oxidations

The effect of hydrophilic ionic liquids (ILs) on the activity of chloroperoxidase (CPO) was checked through kinetic and stereochemical studies. The possibility to employ this enzyme in synthesis has been demonstrated investigating the chemo- and stereoselectivity of oxidation of phenyl methylsulfide in several citrate buffer-IL mixtures.     

New phenolic constituents from the fruit juice of Phyllanthus emblica

Six new phenolic constituents, L-malic acid 2-O- (1), mucic acid 2-O- (5), mucic acid 1,4-lactone 2-O- (6), 5-O- (8), 3-O- (10), and 3,5-di-O- (11) gallates, were isolated from the fruit juice of Phyllanthus emblica together with their methyl esters (2-4, 7, 9), and their structures were determined by spectral and chemical methods. Compounds 5, 6, and 8, the major phenolic constituents of the juice, were present as an equilibrium mixture in aqueous solution.