Alkane Hydroxylation Research Articles

ChemInform Abstract: Mild and Efficient Alkene Epoxidation and Alkane Hydroxylation by Manganese(III) Porphyrin Supported on IRA-400 Ion-Exchange Resin.

Sulfonated manganese(III) tetraphenylporphyrin supported on Amberlite IRA-400 ion-exchange resin (MnTPPS-Ad), can act as an efficient heterogeneous catalyst for alkene epoxidation and alkane hydroxylation by sodium periodate in the presence of imadazole.

The Valence Bond Way: Reactivity Patterns of Cytochrome P450 Enzymes and Synthetic Analogs

The preceding decade has witnessed an immense surge of activity in the bioinorganic chemistry of transition metal enzymes and synthetic analogs that model their operation. The wide range of research covers both experimental and theoretical investigations of structure and reactivity patterns. Theory, and especially density functional theory (DFT), has become a very useful tool, an important partner of experiment in resolving structural and mechanistic issues. This flare of activity has generated a great deal of knowledge on intermediates, transition states, barriers, rate constants, rate-equilibrium relationships, stereoselectivity, and so forth. This abundance of acquired knowledge has created the need for establishing order, namely, the outlining of broad generalizations, as well as the creation of a more-intuitive interface between experimental and theoretical data. The valence bond (VB) diagram model, originally developed for organic reactions, is such a theoretical framework that has the potential to guide the requisite generalizations in the field of bioinorganic chemical reactivity. In this Account, we briefly describe the principles of construction of VB diagrams for bioinorganic reactions, detailing applications in the booming research area of heme enzyme (specifically cytochrome P450) reactivity, and particularly two archetypal reactions of these enzymes, alkane hydroxylation and thioether sulfoxidation. For congruence with the lingua franca of bioinorganic chemistry, the VB model is formulated to create bridges to (i) the molecular orbital (MO) description, (ii) the oxidation state formulation of transition metal complexes, and (iii) widely used concepts such as the Bell-Evans-Polanyi (BEP) principle. The VB diagram model reveals the origins of the barrier, describes the formation of transition states and reaction intermediates, and allows the prediction of barrier heights and structure-reactivity relationships. Thus, from the VB diagram model, we can rationalize the mechanistic selection during alkane hydroxylation compared with thioether sulfoxidation, as well as the different behaviors of the spin states during the reactions with the active species of P450, the high-valent iron oxo species called compound I (Cpd I). Furthermore, the VB model leads to expressions that enable us to estimate barrier heights from easily accessible reactant properties, such as bond energies, ionization potential, and electron affinities. We further show that the model is not limited to these archetypal processes: its applicability is wider and more general. Accordingly, we outline the potential applications of these principles to other reactions of P450 (such as olefin epoxidation and arene hydroxylation) and to similar reactions of nonheme enzymes and synthetic models. The VB diagram model leads to a unified understanding of complex bioinorganic transformations, creates order in the data, and provides an important framework for making useful predictions.

Efficient stereo- and regioselective hydroxylation of alkanes catalysed by a bulky polyoxometalate

Direct functionalization of alkanes by oxidation of C-H bonds to form alcohols under mild conditions is a challenge for synthetic chemistry. Most alkanes contain a large number of C-H bonds that present difficulties for selectivity, and the oxidants employed often result in overoxidation. Here we describe a divanadium-substituted phosphotungstate that catalyses the stereo- and regioselective hydroxylation of alkanes with hydrogen peroxide as the sole oxidant. Both cyclic and acyclic alkanes were oxidized to form alcohols with greater than 96% selectivity. The bulky polyoxometalate framework of the catalyst results in an unusual selectivity that can lead to the oxidation of secondary rather than the weaker tertiary C-H bonds. The catalyst also avoids wasteful decomposition of the stoichiometric oxidant, which can result in the production of hydroxyl radicals and lead to non-selective oxidation and overoxidation of the desired products.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Selective hydroxylation of alkanes catalyzed by iron(IV)corrole

The complex meso-tris(pentafluorophenyl)corrolatoiron(IV)chloride [(F 15TPC)FeCl] emerged as efficient catalyst in hydroxylating alkanes at room temperature. Cyclohexane and adamantane have been oxidized to the corresponding alcohols using m-chloroperbenzoic acid ( m-CPBA) as terminal oxidant. Cyclohexane has been converted to cyclohexanol in 50% yield with 100% selectivity. Adamantane has also been hydroxylated up to 75% overall yield under identical reaction condition. Significantly high regioselectivity in adamantane oxidation has been observed. The reactive intermediates have been quantitatively trapped by 2,4,6-tri- t-butylphenol (TTBP). Kinetic analysis of the (F 15TPC)FeCl-catalyzed oxidation of TTBP has found consistent with rapid reaction of organic substrate with an intermediate formed in the first and rate-determining step.

Valence bond modelling and density functional theory calculations of reactivity and mechanism of cytochrome P450 enzymes: thioether sulfoxidation

This paper introduces valence bond (VB) modelling of the mechanism and reactivity patterns of thioether sulfoxidation by cytochrome P450 enzymes (P450), as observed experimentally and as found computationally using DFT calculations on P450 sulfoxidation of para-(MeO, Me, H, NO2) substituted thioanisoles. Thus, this study addresses the fundamental factors that cause the following two mechanistic puzzles: (a) Why do sulfoxidation reactions by P450 prefer direct oxygen atom transfer, while at the same time, the observed reaction rates exhibit correlations with electron transfer properties, e.g. the redox potentials of the thioethers? (b) Why do these reactions exhibit spin-selective chemistry and proceed on the low spin doublet state, unlike alkane hydroxylation that proceeds via two-state reactivity on both doublet and quartet states? The first puzzle is reminiscent of the classical electron transfer–polar dichotomy in physical organic chemistry, while the second one is unique to the oxidative chemistry by metal–oxo reagents. The VB modelling shows that the doublet state sulfoxidation involves bond formation coupled electron transfer (BFCET), while the quartet state process involves, in addition, a coupled d-orbital excitation, thereby generating a higher barrier. Combined with the previous VB modelling of alkane hydroxylation by P450, the VB diagram model emerges as a conceptual tool that enables understanding of mechanistic puzzles and is capable of estimating barriers in a complex reaction system.

Enhanced Reactivities of Iron(IV)‐Oxo Porphyrin π‐Cation Radicals in Oxygenation Reactions by Electron‐Donating Axial Ligands

The proximal axial ligand in heme iron enzymes plays an important role in tuning the reactivities of iron(IV)-oxo porphyrin pi-cation radicals in oxidation reactions. The present study reports the effects of axial ligands in olefin epoxidation, aromatic hydroxylation, alcohol oxidation, and alkane hydroxylation, by [(tmp)(+*) Fe(IV)(O)(p-Y-PyO)](+) (1-Y) (tmp = meso-tetramesitylporphyrin, p-Y-PyO = para-substituted pyridine N-oxides, and Y = OCH(3), CH(3), H, Cl). In all of the oxidation reactions, the reactivities of 1-Y are found to follow the order 1-OCH(3) > 1-CH(3) > 1-H > 1-Cl; negative Hammett rho values of -1.4 to -2.7 were obtained by plotting the reaction rates against the sigma(p) values of the substituents of p-Y-PyO. These results, as well as previous ones on the effect of anionic nucleophiles, show that iron(IV)-oxo porphyrin pi-cation radicals bearing electron-donating axial ligands are more reactive in oxo-transfer and hydrogen-atom abstraction reactions. These results are counterintuitive since iron(IV)-oxo porphyrin pi-cation radicals are electrophilic species. Theoretical calculations of anionic and neutral ligands reproduced the counterintuitive experimental findings and elucidated the root cause of the axial ligand effects. Thus, in the case of anionic ligands, as the ligand becomes a better electron donor, it strengthens the FeO-H bond and thereby enhances its H-abstraction activity. In addition, it weakens the Fe=O bond and encourages oxo-transfer reactivity. Both are Bell-Evans-Polanyi effects, however, in a series of neutral ligands like p-Y-PyO, there is a relatively weak trend that appears to originate in two-state reactivity (TSR). This combination of experiment and theory enabled us to elucidate the factors that control the reactivity patterns of iron(IV)-oxo porphyrin pi-cation radicals in oxidation reactions and to resolve an enigmatic and fundamental problem.

Synthesis, Structure and Spectral, and Electrochemical Properties of New Mononuclear Ruthenium(III) Complexes of Tris[(benzimidazol‐2‐yl)methyl]amine: Role of Steric Hindrance in Tuning the Catalytic Oxidation Activity

AbstractThe new mononuclear RuIII complexes [Ru(ntb)Cl2]Cl (1), [Ru(ntb)Cl2]ClO4 (1a), and [Ru(mntb)Cl2]Cl (2) (ntb = tris(benzimidazol‐2‐ylmethyl)amine, mntb = tris(N‐methylbenzimidazol‐2‐ylmethyl)amine) have been synthesized and characterized. The X‐ray crystal structure of 1 reveals that the coordination geometry around the RuIII center is distorted octahedral in which four sites are occupied by the tetradentate ligand ntb and the remaining cis positions by two chloride ions. The stronger Ru–Nbzim bonds elongate the Ru–Cl bonds thereby labilizing the coordinated chloride ions. In the electronic absorption spectra the RuIII complexes show two bands corresponding to π(bzim) → t2g(Ru) and pπ(Cl–) → t2g(Ru) ligand‐to‐metal charge transfer (LMCT) transitions along with intraligand transitions in the UV region. Complex 1 shows rhombic EPR spectral features (g1, 2.238; g2, 2.071; g3, 1.790). The RuIII complexes display both RuIII → RuII reduction and RuIII → RuIV oxidation processes. Complexes 1 and 2 catalyze the allylic oxidation of cyclohexene, selective and higher epoxidation of cyclooctene, and hydroxylation of alkanes in the presence of the peroxide TBHP and the peracid m‐CPBA as cooxidants illustrating that the electronic and steric effects of tripodal 4N ligands can be tuned to catalyze the effective oxidative transformation of organic compounds. ESI‐MS studies reveal the formation of ruthenium peroxido species in these catalytic reactions.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Stereospecific CH Oxidation with H2O2 Catalyzed by a Chemically Robust Site‐Isolated Iron Catalyst

Check for cavities: An exceptionally active nonheme iron catalyst employs H2O2 as an oxidant for the stereospecific hydroxylation of alkanes (see scheme). The iron site is located in a chemically robust cavity made up by the ligands.

Metabolic studies of tetrazepam based on electrochemical simulation in comparison to in vivo and in vitro methods

During the last 2 years, the knowledge on the metabolic pathway of tetrazepam, a muscle relaxant drug, was expanded by the fact that diazepam was identified as a degradation product of tetrazepam. The present study demonstrates that this metabolic conversion, recently discovered by in vivo studies, can also be predicted on the basis of a purely instrumental method, consisting of an electrochemical cell (EC) coupled to online liquid chromatography (LC) and mass spectrometry (MS). By implementing a new electrochemical cell type into the EC-LC–MS set-up and by an enhanced oxidation potential range up to 2 V, one limitation of the electrochemical metabolism simulation, the hydroxylation of alkanes and alkenes, has been overcome. Instead of commonly used flow-through cell with a porous glassy carbon working electrode, a wall-jet cell with exchangeable electrode material was used for this study. Thereby, the entire metabolic pathway of tetrazepam, in particular including the hydroxylation of the tetrazepam cyclohexenyl moiety, was simulated. The electrochemical results were not only compared to microsomal incubations, but also to in vivo experiments, by analysing urine samples from a patient after tetrazepam delivery. For structure elucidation of the detected metabolites, MS/MS experiments were performed. The comparison of electrochemistry to in vitro as well as to in vivo experiments underlines the high potential of electrochemistry as a fast screening tool in the prediction of metabolic transformations in drug development.

Highly selective hydroxylation of alkanes catalyzed by (μ-oxo)bis(μ-carboxylato)-bridged diiron(iii) complexes: involvement of mononuclear iron(iii) species in catalysis

A few diiron(III) complexes [Fe2(O)(OAc)2(L1)2](ClO4)2, [Fe2(O)(OBz)2(L1)2](ClO4)2, [Fe2(O)(OAc)2(L2)2](ClO4)2 and [Fe2(O)(OBz)2(L2)2](ClO4)2, where L1=N,N-bis(pyrid-2-ylmethyl)-iso-butylamine, L2=N,N-bis(pyrid-2-ylmethyl)benzylamine, AcO=acetate and BzO=benzoate, have been isolated and characterized by means of elemental analysis and spectral and electrochemical methods. The molecular structures of the complexes and have been determined by single-crystal X-ray diffraction analysis and they possess a distorted bioctahedral geometry in which each iron atom is coordinated to the oxygen atom of the micro-oxo bridge, two oxygen atoms of the micro-benzoato bridges and three nitrogen atoms of L1 and L2 ligands capping the two ends of the diiron(III) cluster. The ESI-MS spectral data of the complexes reveal that the complexes remain intact in dichloromethane (DCM) solution. Upon adding one equivalent of Et3N to a mixture of one equivalent of the diiron(III) complexes and excess of m-chloroperbenzoic acid (m-CPBA) in DCM, an intense absorption band (lambdamax, 670-700 nm) appears, which corresponds to the species [Fe2(O)(OAc)(m-CPBA)(L)2]2+ (ESI-MS, m/z 466) suggested as the intermediate involved in the oxygenation reactions. All the present complexes show efficient alkane hydroxylation with 300-400 turn over numbers and good selectivities for cyclohexane (A/K, 10-14) and adamantane (3 degrees/2 degrees, 9-11). Interestingly, the formation of monoiron(III) species has been discerned in the alkane hydroxylation reactions beyond approximately 50 turnovers. The mononuclear 1:1 iron(III) complexes of L1 and L2 ligands generated in situ are also found to catalyze the oxygenation reactions with high selectivity and efficiency for cyclohexane (A/K, 10-14). Upon their reaction with m-CPBA in DCM, a characteristic absorption band (lambdamax, 600 nm, epsilonmax, 355 M(-1) cm(-1)) appears and decays at room temperature. This spectral feature is consistent with the mononuclear high-valent iron-oxo species suggested as an intermediate in the oxygenation reactions.

Alkane Hydroxylation by Peroxy Acids: A Comparison with the Cytochrome P450 Hydroxylation

Alkane hydroxylation by peroxy acids proceeds by a synchronous nonconcerted peroxy oxygen insertion into the C-H bond according to density functional theory. A comparable reaction sequence, initiated by homolytic peroxy bond cleavage, can be formulated for the alkane hydroxylation by the cytochrome P450 hydroperoxo-heme Compound 0. This hydroxylation reaction proceeds by a two-step process because the formed reactive intermediate, Compound II, is significantly stabilized.

Effect of bulky substitution on catalytic activity of a manganese salen complex used in biomimetic alkene epoxidation and alkane hydroxylation with sodium periodate

The catalytic activity of Mn(salen)Cl containing tert-pentyl groups at the 3,5-positions of the salen ligand in the epoxidation of alkenes and hydroxylation of alkanes was studied at room temperature, using sodium periodate as an oxygen source. The effects of various axial ligands were investigated in the epoxidation of cyclooctene. Imidazole, as a strong π-donor ligand, was the best axial ligand. The effect of different solvents was studied in the epoxidation of cyclooctene in CH3CN/H2O solvent mixture. The epoxidation reactions of cyclooctene by different oxygen donors including NaIO4, Bu4NIO4, KHSO5, H2O2, H2O2/urea, NaOCl and tert-BuOOH were also studied and NaIO4 was selected as oxygen source. The presence of bulky substituents in the 3,5-positions of the salen ligand was found to increase the catalytic activity of this complex.

A Valence Bond Modeling of Trends in Hydrogen Abstraction Barriers and Transition States of Hydroxylation Reactions Catalyzed by Cytochrome P450 Enzymes

The paper outlines the fundamental factors that govern the mechanisms of alkane hydroxylation by cytochrome P450 and the corresponding barrier heights during the hydrogen abstraction and radical rebound steps of the process. This is done by a combination of density functional theory calculations for 11 alkanes and valence bond (VB) modeling of the results. The energy profiles and transition states for the various steps are reconstructed using VB diagrams (Shaik, S. S. J. Am. Chem. Soc. 1981, 103, 3692-3701. Shaik, S.; Shurki, A. Angew. Chem. Int. Ed. 1999, 38, 586-625.) and the DFT barriers are reproduced by the VB model from raw data based on C-H bond energies. The model explains a variety of other features of P450 hydroxylations: (a) the nature of the polar effect during hydrogen abstraction, (b) the difference between the activation mechanisms leading to the Fe(IV) vs the Fe(III) electromers, (c) the difference between the gas phase and the enzymatic reaction, and (d) the dependence of the rebound barrier on the spin state. The VB mechanism shows that the active species of the enzyme performs a complex reaction that involves multiple bond making and breakage mechanisms by utilizing an intermediate VB structure that cuts through the high barrier of the principal transformation between reactants and products, thereby mediating the process at a low energy cost. The correlations derived in this paper create order and organize the data for a process of a complex and important enzyme. This treatment can be generalized to the reactivity patterns of nonheme systems and synthetic iron-oxo porphyrin reagents.

Controlled Oxidation of Hydrocarbons by the Membrane-Bound Methane Monooxygenase: The Case for a Tricopper Cluster

[Figure: see text]. The growing need for inexpensive methods to convert methane to methanol has sparked considerable interest in methods that catalyze this process. The integral membrane protein particulate methane monooxygenase (pMMO) mediates the facile conversion of methane to methanol in methanotrophic bacteria. Most evidence indicates that pMMO is a multicopper enzyme, and these copper ions support redox, dioxygen, and oxo-transfer chemistry. However, the exact identity of the copper species that mediates the oxo-transfer chemistry remains an area of intense debate. This highly complex enzyme is notoriously difficult to purify because of its instability outside the lipid bilayer and tendency to lose its essential metal cofactors. For this reason, pMMO has resisted both initial identification and subsequent isolation and purification for biochemical and biophysical characterization. In this Account, we describe evidence that pMMO is a multicopper protein. Its unique trinuclear copper cluster mediates dioxygen chemistry and O-atom transfer during alkane hydroxylation. Although a recent crystal structure did not show this tricopper cluster, we provide compelling evidence for such a cluster through redox potentiometry and EPR experiments on the "holo" enzyme in pMMO-enriched membranes. We also identify a site in the structure of pMMO that could accommodate this cluster. A hydrophobic pocket capable of harboring pentane, the enzyme's largest known substrate, lies adjacent to this site. In addition, we have designed and synthesized model tricopper clusters to provide further chemical evidence that a tricopper cluster mediates the enzyme's oxo-transfer chemistry. These biomimetic models exhibit similar spectroscopic properties and chemical reactivity to the putative tricopper cluster in pMMO. Based on computational analysis using density functional theory (DFT), triangular tricopper clusters are capable of harnessing a "singlet oxene" upon activation by dioxygen. An oxygen atom is then inserted via a concerted process into the C-H bond of an alkane in the transition state during hydroxylation. The turnover frequency and kinetic isotope effect predicted by DFT show excellent agreement with experimental data.

Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB

AlkB stops the radical clock: Three structurally analogous radical-clock substrates with a large span in their rearrangement rates are hydroxylated by AlkB to afford similar amounts of rearranged (2) and unrearranged products (1). Such a result is in accord with radical rebound competing with cage escape of the geminate substrate radical. The results show that radical clocks can measure both the radical lifetime and the kinetics of cage escape.

Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB

AlkB hält die Radikaluhr an: Drei strukturell ähnliche Radikaluhr-Substrate mit sehr unterschiedlichen Umlagerungsgeschwindigkeiten liefern bei der Hydroxylierung durch AlkB umgelagerte (2) und nichtumgelagerte Produkte (1) in ähnlichen Mengen. Dieser Befund ist in Einklang mit der Konkurrenz zwischen einem „Radical rebound“ des Substratradikals und seinem Entkommen aus dem Käfig und belegt, dass Radikaluhren sowohl die Lebensdauer des Radikals als auch die Kinetik des Verlassens des Käfigs messen können. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2008/z801184_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Diatomite-supported manganese Schiff base: An efficient catalyst for oxidation of hydrocarbons

The manganese(III)-salophen complex has been successfully immobilized for the first time on natural diatomite by a simple procedure. This catalyst was characterized by FT-IR, UV–vis, SEM and elemental analysis. The new catalytic material has been evaluated as oxidation catalyst. Our results show that these composite exhibit significant catalytic activities for alkene epoxidation and alkane hydroxylation using sodium periodate as oxidant.

Probing the Hydrophobic Pocket of the Active Site in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by Variable Stereoselective Alkane Hydroxylation and Olefin Epoxidation

pMMO from M. capsulatus (Bath) oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L-[2-(2)H(1),3-(2)H(1)]butane, the reactions proceed through the concerted O-atom insertion mechanism. However, when propene and but-1-ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans-but-2-ene, which reveal only the D,L-2,3-dimethyloxirane products, and on cis-but-2-ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3-trifluoroprop-1-ene as the substrate. The products obtained are 90 % (2S)-oxirane. When 1,1,1-trifluoropropane is the substrate, the hydroxylation at the 2-carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF(3) group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme.

Silica supported Mn(Br 8TPP)Cl and Mn(TPP)Cl as efficient and reusable catalysts for selective hydrocarbon oxidation under various reaction conditions: The effect of substituted bromines on the catalytic activity and reusability

The imidazole-modified silica (SiIm) was used for immobilization of octabromotetraphenylporphyrinatomanganese(III) chloride, Mn(Br 8TPP)Cl and tetraphenylporphyrinatomanganese(III) chloride, Mn(TPP)Cl. SiIm is not only a heterogeneous axial base but also a support for immobilization of manganese porphyrins. [Mn(Br 8TPP)Cl-SiIm] and [Mn(TPP)Cl-SiIm] as biomimetic oxidation catalysts catalyze alkene epoxidation and alkane hydroxylation with NaIO 4 under agitation with magnetic stirring. Ultrasonic irradiation enhanced the catalytic activity of these catalysts in alkene epoxidation and alkane hydroxylation and this led to shorter reaction times and higher product yields. These new heterogenized catalysts could be reused several times without significant loss of their catalytic activity.