Formation of iron(II)-nitrosoalkane complexes: a new activity of microperoxidase 8

Microperoxidase 8 (MP8) is a heme octapeptide, obtained by enzymatic hydrolysis of heart cytochrome c, in which a histidine is axially coordinated to the heme iron, and acts as its fifth ligand. It exhibits two kinds of activities: a peroxidase-like activity and a cytochrome P450-like activity. We here show that MP8 is not only able to oxidize various aliphatic and aromatic hydroxylamines with the formation of MP8-Fe(II)-nitrosoalkane or -arene complexes absorbing around 414 nm, but also that these complexes can be obtained by reduction of nitroalkanes. This is the first example of fully characterized iron(II)-metabolite complexes of MP8. Such complexes constitute good models for those obtained upon oxidation of amphetamine or macrolids by cytochromes P450. In addition, this is a new catalytic activity of MP8, which validates the use of this mini-enzyme as a convenient model for hemoproteins of interest in toxicology and pharmacology such as cytochromes P450 and peroxidases.     

Biocatalytic and biomimetic oxidations with vanadium

Approaches to the rational design of vanadium-based semi-synthetic enzymes and biomimetic models as catalysts for enantioselective oxidations are reviewed. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which in vivo mediates the hydrolysis of phosphate esters, afforded a semi-synthetic peroxidase. It catalyzed the enantioselective oxidation of prochiral sulfides with H2O2 affording the S-sulfoxide, e.g. in 66% ee at quantitative conversion of thioanisole. Under the reaction conditions the semi-synthetic vanadium peroxidase was stable for more than 3 days with only a slight decrease in turnover frequency. Amongst the transition-metal oxoanions that are known to be potent inhibitors of phosphatases, only vanadate resulted in a semi-synthetic peroxidase when incorporated into phytase. In a biomimetic approach, vanadium complexes of chiral Schiff base complexes were encapsulated in the super cages of a hydrophobic zeolite Y. Unfortunately, these ship-in-a-bottle complexes afforded only racemic sulfoxide in the catalytic oxidation of thioanisole with H2O2.     

Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines

Various N-alkylhydroxylamines such as N-hydroxyamphetamine react with prostaglandin synthase (PGHS) from sheep seminal vesicles, with the formation of new complexes characterized by a Soret peak around 421 nm. These complexes are very stable toward O2 or dithionite but are destroyed upon oxidation by Fe(CN)6K3 with regeneration of starting PGHS-FeIII. Their spectral characteristics, chemical properties, and routes of formation (either by direct oxidation of RNHOH or by in situ reduction of RNO2 in the presence of dithionite) are very similar to those previously reported for nitrosoalkane complexes of hemoglobin-, myoglobin-, and cytochrome P-450-FeII. Their FeII-N(O)R structure was completely confirmed in the case of N-hydroxyamphetamine, both by extraction of the heme complex by butanone and by identification to authentic protoporphyrin IX-FeII-N(O)-amphetamine, and by insertion of this authentic complex into apoPGHS. Phenylhydroxylamine also reacts with PGHS-FeIII to give a PGHS-FeII-N(O)Ph complex which is not stable in the presence of dithionite because of its weaker PGHS-FeII-N(O)R bond when compared to PGHS-FeII-nitrosoalkane complexes. The ability of various N-alkylhydroxylamines to form PGHS-FeII-N(O)R complexes greatly depends upon their hydrophobicity. Actually, CH3NHOH and C2H5NHOH are totally inactive whereas about 10 molar excess of N-hydroxyamphetamine and C6H5NHOH already lead to 50% complex formation. This is in favor of an hydrophobic environment of the heme in PGHS. Finally, PGHS engaged in such FeII-nitrosoalkane complexes completely loses its dioxygenase activity, suggesting that N-substituted hydroxylamines or compounds that can be metabolized in vivo to give such hydroxylamines could act as strong PGHS inhibitors.     

Regioselective nitration of phenol induced by catalytic antibodies

Catalytic antibodies with a metalloporphyrin cofactor represent a new generation of biocatalysts tailored for selective oxidations. Thus monoclonal antibodies, 3A3, were raised against microperoxidase 8 (MP8), and the corresponding 3A3-MP8 complexes were shown previously to have a high peroxidase activity. This paper shows that those complexes also catalyzed efficiently the nitration of phenol into 2- and 4-nitrophenol by NO₂ ^– in the presence of H₂O₂. pH dependence studies suggested that no amino acid from the antibody protein participated in the heterolytic cleavage of the O-O bond of H₂O₂. The inhibition of the reaction by cyanide and radical scavengers suggested a MP8-mediated peroxidase-like mechanism, involving the reduction of high-valent iron-oxo species by NO₂ ^– and phenol producing, respectively, NO₂ · and phenoxy radicals, which then reacted to give nitrophenols. Finally, the antibody protein appears to have two major roles: (i) it protects MP8 toward oxidative degradations and (ii) it induces a regioselectivity of the reaction toward the formation of 2-nitrophenol.     

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.     

Enantioselective oxidations of sulfides catalyzed by chloroperoxidase

The chloroperoxidase-catalyzed and horseradish peroxidase catalyzed oxidations of sulfides by tert-butyl and other peroxides have been investigated. The former metal enzyme afforded the corresponding sulfoxides having R absolute configuration in up to 92% enantiomeric excess (ee), whereas the latter gave racemic products. The various factors that control the enantioselectivity of the oxygenation have been examined.     

Peroxidase-catalyzed asymmetric sulfoxidation in organic solvents versus in water

Peroxidase-catalyzed asymmetric sulfoxidations, while synthetically attractive, suffer from relatively low reaction rates due to poor substrate solubilities in water and from appreciable spontaneous oxidation of substrates (especially aryl alkyl sulfides) with H(2)O(2). In this work, we found that both of these shortcomings could be alleviated by switching from aqueous solutions to certain nearly anhydrous (99.7%) organic solvents as sulfoxidation reaction media. The rates of spontaneous oxidation of the model prochiral substrate thioanisole in several organic solvents were observed to be some 100- to 1000-fold slower than in water. In addition, the rates of asymmetric sulfoxidation of thioanisole in isopropyl alcohol and in methanol catalyzed by horseradish peroxidase (HRP) were determined to be tens to hundreds of times faster than in water under otherwise identical conditions. This dramatic activation is due to a much higher substrate solubility in organic solvents than in water and occurs even though the intrinsic reactivity of HRP in isopropyl alcohol and in methanol is hundreds of times lower than in water. Sulfoxidation of thioanisole catalyzed by four other hemoproteins (soybean peroxidase, myoglobin, hemoglobin, and cytochrome c) is also much faster in isopropyl alcohol than in water.     

Enantiomeric and mesomeric mandelate complexes of molybdenum -- on their stereospecific formations and absolute configurations

The stereospecific formation and absolute configuration of R-homocitrate coordinated FeMo-co in nitrogenase was mimicked through the structural analyses of a collection of enantiomeric and mesomeric mandelato molybdenum complexes, i.e., (NH(4))(2)[Mo(Delta)O(2)(R-mand)(2)]x3H(2)O (1a), (NH(4))(2)[Mo(Lambda)O(2)(S-mand)(2)]x3H(2)O (1b), (NH(4))(4)[Mo(Delta)O(2)(RS-mand)(2)][Mo(Lambda)O(2)(RS-mand)(2)]x8H(2)O (2), (NH(4))(2)[W(Delta)O(2)(R-mand)(2)]x2H(2)O (3a), (NH(4))(2)[W(Lambda)O(2)(S-mand)(2)]x2H(2)O (3b) (H(2)mand=mandelic acid, C(8)H(8)O(3)), which have been characterized by elemental analyses, optical rotation, circular dichroism, IR, NMR spectroscopes and X-ray single crystal studies. The R and S chiral mandelic acids induce the formations of the enantiomeric pair of chiral complexes, which are supported by the characterizations of optical rotation and circular dichroism. The configuration of the resulted metal center could be assigned as Delta or Lambda. While the RS racemic reagent yields only mesomeric compound. The Delta(R,R)-complexes 1a and 3a are enantiomers of Lambda(S,S)-1b and 3b, respectively. Of the five complexes, Mo and W atoms are all hexa-coordinated by two cis-oxo groups and two bidentate mandelate ligands through the deprotonated alpha-alkoxyl and alpha-carboxyl groups, forming a stable five-membered chelated rings. The average Mo(VI)-O bond distances with alpha-alkoxyl and alpha-carboxyl are 1.944 and 2.210 A, respectively. Further comparison indicates that bonds of alpha-alkoxyl groups in the hydroxycarboxylato molybdenum complexes are much sensitive to the change in the oxidation state of molybdenum, which support the possible Mo activation model in FeMo-co through the protonation and cleavage of alpha-alkoxyl group in homocitrate ligand.     

Chloroperoxidase-catalyzed enantioselective oxidations in hydrophobic organic media.

Chloroperoxidase from Caldariomyces fumago, a peroxidase that performs P450-like chemistry, was immobilized via covalent attachment into polyurethane foam as well as conjugated with a surfactant or polymer via colyophilization. The resulting preparations catalyzed enantio- and regioselective oxidations in hydrophobic organic media with tert-butyl hydroperoxide as the oxidant.Dried PUR-foam immobilized CPO mediated the selective oxidation of indole to 2-oxindole (regioselectivity: 99%) in water-saturated isooctane or 1-octanol. Thioanisole was converted into the corresponding (R)-sulfoxide (ee > 99%) in isooctane medium.The complexes of CPO with sodium octadecylsulphate or ethyl cellulose mediated the oxidation of thioanisole in water-immiscible organic media with variable enantioselectivity due to radical side-reactions. In the presence of alpha-tocopherol, acting as radical scavenger, the (R)-sulfoxide was formed with ee > 90%. The effect of the water activity on the catalytic activity of the complexes was investigated.The CPO complexes likewise mediated the regioselective oxidation of indole into 2-oxindole in water-saturated isooctane or 1-octanol and its kinetics were investigated. The reaction suffered from substrate inhibition when carried out in isooctane.     

Tyrosinase catalyzes asymmetric sulfoxidation

Mushroom tyrosinase was found to catalyze the oxidation of organic sulfides to sulfoxides in the presence of a catechol as cosubstrate, in a reaction which is unprecedented for this enzyme and resembles those performed by external monooxygenases. Only the oxy form of the enzyme is in fact capable of oxidizing the sulfide in a two-electron process, while the resulting met form can only be recycled by reduction with catechol. The cosubstrate competes with the sulfide also in the reaction with oxy-tyrosinase. For this reason, the sulfoxidation of thioanisole in the presence of l-3,4-dihydroxyphenylalanine (L-dopa) occurs with moderate yields ( approximately 20%) but high enantioselectivity ( approximately 85% e.e.), and favors ( S)-methyl phenyl sulfoxide. The enantioselectivity can be further increased to >90% when excess ascorbic acid is added to the reaction to limit enzyme inactivation by the quinones produced by L-dopa oxidation. An experiment using (18)O 2 showed that 18-O incorporation into methyl phenyl sulfoxide was above 95%, confirming that the mechanism of the sulfoxidation involves oxygen transfer from oxy-tyrosinase to the sulfide.     

Spectroscopic investigation of isonitrile complexes of ferric and ferrous microperoxidase 8

Microperoxidase 8 (MP8) is able to react with alkyl- and aryl-isonitriles (RNC) both in its reduced and oxidized states, to form MP8Fe(II)- and MP8Fe(III)-CNR complexes. The coordination and spin states of these complexes have been fully characterized by UV-visible and resonance Raman spectroscopies. Both MP8Fe(II)- and MP8Fe(III)-CNR complexes are hexacoordinate low-spin complexes, which bear a single RNC ligand on the distal face of the heme and keep the His 18 ligand on its proximal face, trans to the RNC ligand. A comparison of these characteristics with those of the Fe-CNR complexes of other hemoproteins suggests that both MP8Fe(II)- and MP8Fe(III)-CNR complexes present a Fe-C-N linear arrangement. This may be due to the lack of any interactions of the RNC ligand with the octapeptide of MP8 that is mainly located over the opposite face of the heme. Finally the formation of hexacoordinate low-spin MP8Fe(II)- and MP8Fe(III)-CNR complexes constitutes a new example of the reactivity of MP8 with a new class of weak sigma-donating and strong pi-accepting ligands, which adds to its already very rich coordination chemistry.     

One-pot synthesis and chiral analysis of cyclopropane derivatives

A user-friendly, one-pot procedure was developed to access racemic as well as enantiomerically enriched cyclopropanes. Thus, the cyclopropanation of olefin (3) was performed using Meldrum's acid (4) or dimethyl malonate (5) and diacetoxyiodobenzene PhI(OAc)2 (6) or iodosyl benzene PhI=O (7) for in situ generation and decomposition of the phenyliodonium ylide 1 and 2, respectively. The reaction proceeds well with 5 mol% of achiral rhodium (II)-catalyst [Rh2(OAc)4] and a 10-fold excess of olefin affording the cyclopropane derivates 10 and 11, respectively, with high yield. The system is compatible with chiral Rh(II)-catalysts 8 and 9 and an enantiomeric excess up to 66% was achieved. An effective baseline separation of the enantiomers of the resulting cyclopropane derivatives was achieved using gas chromatography on the chiral stationary phase Chirasil-beta-dex.     

Synthesis and biological evaluation of copper (II) complexes of sterically hindered o-aminophenol derivatives as antimicrobial agents

Cu(II) complexes with 4,6-di(tert-butyl)-2-aminophenol (I) and 2-anilino-4,6-di(tert-butyl)phenol (II) have been synthesized and characterized by means of elemental analysis, TG/DTA, FT-IR, UV-vis, ESR, and conductance measurements. The compounds I and II can coordinate in their singly deprotonated forms and behave as bidentate O,N-coordinated ligands; their CuL(2) complexes are characterized by CuN(2)O(2) coordination modes and square planar geometry. In vitro antimicrobial screening against Gram-positive and Gram-negative bacteria, yeasts, and moulds indicated that the compound I and its Cu(II) complex were more active than Questiomycin B, the compound II, and its Cu(II) complex.     

Selective oxidation of sulfides to sulfoxides catalyzed by ruthenium (III) meso-tetraphenylporphyrin chloride in the presence of molecular oxygen

Highly efficient selective oxidation of sulfides to sulfoxides by molecular oxygen catalyzed by ruthenium (III) meso-tetraphenylporphyrin chloride (Ru(TPP)Cl) with isobutyraldehyde as oxygen acceptor has been reported. In large-scale experiment of thioanisole oxidation, the isolated yield of sulfoxide of 92% was obtained and the turnover number reached up to 92,000.     

Dirhodium-catalyzed enantioselective C-H insertion of N-(2-benzyloxyethyl)-N-(tert-butyl)diazoacetamide and its application for the synthesis of chiral GABOB

Rh(2)(4S-MEOX)(4) and ethereal solvent are the best catalytic system for the enantioselective intramolecular C-H insertion of N-(2-benzyloxyethyl)-N-(tert-butyl)diazoacetamide 2. The highest enantiomeric excess obtained was 91%. A new route for the asymmetric synthesis of gamma-amino-beta-hydroxybutyric acid (GABOB) has been developed.     

Improved operational stability of peroxidases by coimmobilization with glucose oxidase

The operational stability of peroxidases was considerably enhanced by generating hydrogen peroxide in situ from glucose and oxygen. For example, the total turnover number of microperoxidase-11 in the oxidation of thioanisole was increased sevenfold compared with that obtained with continuous addition of H(2)O(2). Coimmobilization of peroxidases with glucose oxidase into polyurethane foams afforded heterogeneous biocatalysts in which the hydrogen peroxide is formed inside the polymeric matrix from glucose and oxygen. The total turnover number of chloroperoxidase in the oxidation of thioanisole and cis-2-heptene was increased to new maxima of 250. 10(3) and 10. 10(3), respectively, upon coimmobilization with glucose oxidase. Soybean peroxidase, which normally shows only classical peroxidase activity, was transformed into an oxygen-transfer catalyst when coimmobilized with glucose oxidase. The combination catalyst mediated the enantioselective oxidation of thioanisole [50% ee (S)] with 210 catalyst turnovers.     

Spectral studies on the oxidation of organic sulfides (thioanisoles) by horseradish peroxidase compounds I and II

Using both rapid-scan and conventional spectrophotometry, oxygenation of p-substituted thioanisoles by horseradish peroxidase compounds I and II was investigated at pH 5, 7 and 9. The pH-jump technique was applied to the compound II reactions at acidic and neutral pH. The rate of oxidation of the sulfides is dependent on pH, concentration of substrate and on the different substituents in the para position of the benzene ring. Our results, based on transient state observations of the enzyme intermediates, are in agreement with the results of Kobayashi, S., Minoru, N., Kimura, T. and Schaap, A.P. (Biochemistry (1987) 26, 5019-5022), obtained using 18O-labelling and studies of product formation, in which formation of a sulfur cation radical from compound I is proposed. We consider two reaction mechanisms for the compound II reaction: one a one-electron oxidation of the thioanisole, analogous to the compound I reaction, and the other, the attack of the hydroxyl radical originating from compound II on the sulfur-cation radical.     

Covalently modified microperoxidases as heme-peptide models for peroxidases

Microperoxidase-8 (MP8) and microperoxidase-9 (MP9) have been covalently modified by attachment of proline-containing residues to the amino terminal peptide chain in order to obtain new peroxidase model systems. The catalytic activities of these derivatives in the oxidation of p-cresol by hydrogen peroxide have been compared to that of MP8. The presence of steric hindrance above the heme reduces the formation rate of the catalytically active species, while the reactivity is increased when the amino group of a proline residue is close to the iron. The modification of the catalyst affects the rate of degradation processes undergone by the heme group during catalysis. A bulky aromatic group on the distal side decreases the stability of the complex because it reduces the mobility of a phenoxy radical species formed during catalysis, while the presence of proline residues increases the number of turnovers of the heme catalysts before degradation. The complex Pro2-MP8 obtained by addition of two proline residues to MP8 exhibits the best catalytic performance in terms of activity and chemical stability.     

Structural correlation of catecholase-like activities of oxy-bridged dinuclear copper(II) complexes

Eight oxy-bridged dinuclear copper(II) complexes with catecholase-like sites, [Cu(L1)X]2 (HL1 = 1-diethylaminopropan-2-ol, X=N3- 1, NCO- 2, and NO2- 3), [Cu(L2)X]2 (HL2=N-ethylsalicylaldimine, X=NO3- 4, Cl- 5, N3- 6, NCS- 7), and [Cu(L3)]2(ClO4)2, 8 (HL3=N-(salicylidene)-N'-(2-pyridylaldene)propanediamine) have been prepared and characterized. The single crystal X-ray analysis show that the structures of complexes 6 and 8 are dimeric with two adjacent copper(II) atoms bridged by pairs of micro-oxy atoms from the L2 and L3 ligands. Magnetic susceptibility measurements in the temperature range 4-300 K indicate significant antiferromagnetic coupling for 4, 5 and 7 and ferromagnetic coupling for 6 between the copper(II) atoms. The catecholase activity of complexes for the oxidation of 3,5-di-tert-butylcatechol by O2 was studied and it was found that the complexes with the bond distance of Cu(II)...Cu(II) located at 2.9-3.0 A show higher catecholase activity.     

Hydrogen atom abstraction by Cu(II)- and Zn(II)-phenoxyl radical complexes, models for the active form of galactose oxidase

The Cu(II) and Zn(II) complexes of phenoxyl radical species [M(II)(L1*)(NO3)]+ (M=Cu or Zn, L1H: 2-methylthio-4-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol ) and [M(II)(L2*)(NO3)]+ (M=Cu or Zn, L2H: 2,4-di-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol) are prepared as model complexes of the active form of galactose oxidase (GAO). Hydrogen atom abstraction of 1,4-cyclohexadiene and tert-butyl substituted phenols by the GAO model complexes proceeds very efficiently to give benzene and the corresponding phenoxyl radical or its C-C coupling dimer as the oxidation products, respectively. Kinetic analyses on the oxidation reactions have shown that the hydrogen atom abstraction of the phenol substrates is significantly enhanced by the coordinative interaction of the OH group to the metal ion center of the complex, providing valuable insight into the enzymatic mechanism of the alcohol oxidation. Details of the substrate-activation process have been discussed based on the activation parameters (deltaH* and deltaS*) of the reactions.