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UvA-DARE (Digital Academic Repository)

Enantioselective oxygen-transfer reactions catalyzed by peroxidases

Tuynman, A.

Publication date 1999

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Citation for published version (APA): Tuynman, A. (1999). Enantioselective oxygen-transfer reactions catalyzed by peroxidases.

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Download date:29 Sep 2021 Introduction

Chapter 1 Introduction

IOP In order to maintain and to improve the competitive position of the Dutch industries in the world-wide economic scene, the Netherlands Ministry of Economic Affairs promotes innovative research in a number of promising fields. The ultimate goal being "the creation of new economic activity" it has set itself the objective of "providing an essential step on the road from fundamental science to novel applicable technology" [ 1J. Therefore it provides universities and non-profit research institutes with additional subsidiary funding for research projects adapted to the swiftly changing needs of the Dutch industry. Moreover, these programs, called "Innovation Oriented research Programs" (IOP's), are intended to reinforce the collaboration between universities, research institutes and industries. The "Innovative" character is stressed and must be considered from an economical point of view : to make an effort to transform knowledge (science) into know-how (technology). The "technology 5-curve" (Fig. 1) relates the phase of research (in lOP-terms often called "Green Axis") to the applicability or maturity of a technology ("Yellow Axis").

Exploratory Research Applied i DevekjjWflCn't"'^ ! Optimisation Research J ^^^ '

Science i Mission J/ ' • Oriented ; Orienicd J Economic 1 i j evaluation '

Green Ans R&D phase —>-

Figure 1. The "technology S-curve" relating the phase of research (in IOP-terms often called "Green Axis") to the applicability or maturity of a technology ("Yellow Axis").

Thus at the start of a project mainly fundamental knowledge is acquired (science oriented). At a certain stage a mission can be defined to pursue an applicable technology, and the research becomes "Mission Oriented". It is at this hinge that the IOP commences to play a role. The Chapter 1 _____ definition of such mission oriented research targets may constraint the academic researchers (bottom-up) but, at the other hand, ensures a solid support from the industry (top-down).

IOP catalysis 20 % to 30 % of the Gross National Product of the Netherlands is generated by the chemical industry [ 1 ]. Since catalytic conversions are involved in over 80 % of the total chemical production, it may be of no surprise that catalysis is of strategic interest for the Dutch Industry and can be considered as its cornerstone. Also at the science level Dutch universities have a good international reputation in catalysis research and are called "The Dutch School of Catalysis" [2]. The maintenance and development of the knowledge base of catalysis is therefore an imperative and has recently resulted in the institution of a "National Research School of Combination Catalysis".

Central Theme Precision in chemical conversions, in order to save energy and raw materials as well as to avoid the formation of undesired by-products and waste, is the central theme of IOP catalysis. Although catalysis is applied on a large scale in petroleum refining and in the production of bulk chemicals, this is far less the case in the production of fine chemicals. Classical procedures are still applied, using undesirable, toxic and corrosive reagents, that often form by-products and involve low selectivity. Therefore the lOP-catalysis wants to introduce novel catalytic routes in the fine chemical industry.

History and Biocatalysis The first four years of lOP's started in 1989, the two focal points being : "catalyst preparation" and "heterogeneous and homogeneous catalysis". The second 4 year programme was established in 1994. "'Catalyst preparation" was extended to "catalyst preparation and reactor technology" constituting 20 % of the budget, "heterogeneous and homogeneous catalysis" made up 40 % of the budget and a third new programme: "Biocatalysis" consumed the rest of the tripod's budget. Biocatalysis was started as a new catalytic tool to achieve in particular enantioselective synthesis of building blocks for pharmaceuticals and flavours and fragrances. It is in the light of these targets, that the research proposal of the present thesis was aimed. Introduction

Objective The research described in this thesis was part of the research in the IOP clusterproject [3] : "Peroxidases as catalysts in the production of fine chemicals". This clusterproject consisted of five projects, as listed below: A: IKA94002 Peroxidase mediated biotransformations useful in the biocatalytic production of vanillin, carried out by ir.R.ten Have from the Wageningen Agriculture University under supervision of dr J.A.Field and prof.dr.ir.J.A.M.de Bont. B: 1KA94045 Microperoxidases as biocatalysts in regioselective oxygen-transfer reactions with clean oxidants, carried out by ir.M J.M.van Haandel from the Wageningen Agriculture University under supervision of prof.dr.ir.I. M.CM.Rietjens and prof.dr.ir.C.Laane. C: IKA94047 Peroxidases as natural catalysts in the production of (cnantiomerically pure) alcohols and epoxides, carried out by the author of this thesis, under supervision of dr.R.Wever and prof.dr.H.E.Schoemaker. D: IKA94052 Application of vanadium peroxidases as novel biocatalysts, carried out by drs. H.Bien Brink, under supervision of dr.R.Wever and prof.dr.H.E.Schoemaker. E: 1KA94013 Application of redox enzymes in the synthesis of fine chemicals, carried out by ir. F.van de Velde, under supervision of dr.ir. F. van Rant wijk and prof.dr.R.A.Sheldon.

The corollary objective of these project was the development of biocatalysts for the production of fine chemicals via (enantioselcctive) oxidation of industrially accessible starting materials. The first two projects were predominantly focusscd on the development of peroxidases as natural catalysts for the synthesis of flavours and fragrances. The last three projects were aimed at the development of biocatalysts for (enantio)selective oxygen-transfer reactions. An approach of a cluster was chosen to be able to bring together researchers of several complementary disciplines that constitute the broad field of biocatalysis. Thus enzymology, biochemistry, organic chemistry, microbiology and molecular biology were brought together. The objective of the third project and this thesis, has been presented at numerous IOP meetings as : "The development of biocatalysts : (modified) haem peroxidases, useful in the production of chiral epoxides and alcohols" or "Enantioselective oxygen-transfer catalysed by peroxidases". Within the group this specific research was aimed to provide insight into the

3 Chapter 1 mechanistic aspects of these reactions to be able to optimise these conversions with respect to yield and e.e. Whereas other participants had a mission oriented on the application side of the field, this particular research was specifically focussed on the fundamental, scientific knowledge basis of the mechanisms involved to provide support for the other projects in the cluster. Target reactions in this project were the enanlioselective sulphoxidation of thioanisole and the enantioselective epoxidation of styrene to be catalysed by a peroxidase using H202 as a clean oxidant (Fig. 2).

S\5 <\ so <^\ H?02 ^^/ CH CH3 ^2» ITT •**,^ + f ^T 3

peroxidase 'vs^' \^

^> peroxidase (/

Figure 2. Chemical representation of the peroxidase-catalysed (enantioselective) sulphoxidation of thioanisole and the (enantioselective) epoxidation of styrene using H202 as a clean oxidant.

For the enantioselective sulphoxidation of thioanisole to form the (R)-sulphoxide already a peroxidase system existed, that yielded this enantiomer in 99 % e.e. [4], being the chloroperoxidase from Caldariomyces fumago. Provided that the right conditions were chosen a complete conversion can be obtained with this catalyst. Research with a more application oriented character on this enzyme i.e. medium engineering and immobilisation studies was carried out in the fifth project. This enzyme also catalysed the enantioselective epoxidation of styrene, although the (ff)-styreneoxide formed had an e.e. of only 49 % [5]. It must be noted that at the moment the best catalyst in the styrene epoxidation, is a Mn-salen complex (Jacobsen-catalyst) that provides 88 % yield of styreneoxide (86 % e.e. (/?)-enantiomer) at - 78°C [6]. Ergo, at the moment a catalyst docs not yet exist, that produces styreneoxide with an enantiopurity that is industrially interesting. Therefore in this research we focussed to obtain catalysts to produce (S)-sulphoxides and (S)- and (tf)-cpoxides, respectively. Introduction

The catalysis of enantioscleclive sulphoxidation of a sulphide by haem peroxidases has been examined first, since this was commonly believed to be a more simple oxygen-transfer than the epoxidation of an alkene. This reaction would serve as a pioneer or model reaction to obtain favourable conditions for the epoxidation of an alkene and in a later stage of the research, the benzylic hydroxylation of an arylalkanc.

As was mentioned in the justification of these projects, specific topics were investigated by two participants in a joint effort. Such has also been the case for the present thesis: the author also contributed to the project D: Application of vanadium peroxidases as novel biocatalysts, due to considerable overlap in analytic techniques and target reactions that project C and D had in common. This contribution is described in chapter 5.

Chiral epoxides and sulphoxides as chiral synthons, chiral auxiliaries The target molecules of this thesis arc chiral epoxides and chiral sulphoxides. Figure 3 shows the general chemical representation of the two stereoisomers of these molecules, provided that

R] * R2 or R3 * R4. ob V rV R

O R p R4^A-X^ 33 n^llu../ \ ..irtMlA nr R2 *r ^Ri

Figure 3. General chemical representation of Ihe two stereoisomers of sulphoxides and alkenes, when Rj ^ R2 or

R3* R,.

"The epoxide functional group is one of the most useful intermediates in organic synthesis", was the first sentence of a comprehensive review on the "Chemical and biological synthesis of chiral epoxides" [7], Epoxides can undergo stereospecific ring-opening reactions to form Chapter 1 bifunctional compounds (Fig. 4) and also serve as direct chiral building blocks in organic synthesis.

Figure 4. Stereospccific ring-opening reactions of epoxides to form bifunctional compounds. * stereocentre.

Epoxides have been key intermediates in the synthesis of complex optically pure bioactive compounds such as leukotriene, erythromycin, GABOB (y-amino-ß-butyric acid), diltiazem and even as end products with biological activity such as (+)-disparlure (Fig. 5). Thus the development of methods for the synthesis of optically pure epoxides has gained great interest. Sulphoxides are also an important class of Chirons: "Chiral sulphoxides belong to the class of chiral organosulphur compounds which are most widely used in asymmetric synthesis. They are applied as chiral synthons, due to their easy availability and the high asymmetric induction exerted by the chiral sulphinyl group" [8]. Chiral sulphoxides are applied in three areas of synthesis :

1) Reactions of a-sulphinyl carbanions with a broad variety of cleclrophiles.

2) Reactions of oc,ß-unsaturated sulphoxides. 3) Introduction of heteroatomic groups to sulphoxides in their transformation. It would go beyond the scope of this thesis to discuss all these synthetic pathways. Therefore only some prominent applications will be mentioned. Introduction

COOCH,

Erythronolide

Erythromycin

Diltiazem (+)-Disparlure

Figure 5. Epoxides as key intermediates in the synthesis of complex optically pure bioactive compounds.

Ad 1) Dialkyl and alkyl aryl sulphoxides are extremely useful in the introduction of an alkyl chain in a molecule trans to the sulphoxide with complete stereoselectivity. Such has been presented by Marquet et al. in the total synthesis of biotin (9] (Fig. 6). Moreover, threo-1 was converted in the epoxide (+)-disparlurc with 55 % e.e. [10].

H2iC10 H 1)Zn/Me3SiCI CHMe 2) Me30*BF ", NaOH hN-s^V(CH2k 2 4 < r>N' k *\ u r. HO H

The reaction of cc-sulphinyl carbanions with aldehydes and ketones leading to ß- hydroxyalkylsulphoxides is generally highly diastcreoselective and has been used in the synthesis of enantiopure /.voepijuvabiol and eptjuvabiol [11], Two enantiopure sulphoxides 2 and 3 were applied as substrates (Fig. 7). Chapter 1

O 9 MeLi HMPA/THF

Bn—C ^—Bn or HMP/Vdiglyme Bn—f J— Bn i-(CH2)-C02Bu' *- HM'') ("HH 80%

(CH2)4C02Bu' (CH2)4C02Bu'

Figure 6. Chiral sulphoxides as useful intermediates in the total synthesis of biotin.

1)LDA Me OH

S l! 3:2 p-Tol^ >0 >Tor v0

Raney Ni Raney Ni 67% 67%

Me OH Me Me OH

p-Tol- V0

Me02C' Me02C isoepijuvabiol epijuvabiol

Figure 7. Synthesis of enantiopure «oepijuvabiol and epijuvabiol starting from the enantiopure sulphoxides 2 and 3. Each of them was separately dcprotonated and quenched with 3-methyl-butanal to give a mixture of diaslereomeric adducts in a 3:2 ratio. The sequence of reactions of sulphoxide 2 is shown.

Ad 2) The first step in the synthesis of the sequiterpenoid (+)-Hirsutene has been the conjugate addition of the carbanion generated from the sulphoxide to 2-methyl-2- cyclopentenone 112,13] (Fig. 8). Introduction

1) LDA OAc

3) AcCI Tol-p L.rtH SOTol-p

1)Zn/AcOH 2) TiCI4/AcOH

STol-p several steps ió

(+)-Hirsutene

Figure 8. Synthesis of the sequiterpenoid (+)-Hirsutene staring from a sulphoxide.

Also in the synthesis of the anti-tumour activity containing trichothecenes such an addition was a key reaction [14]. Molecules bearing a variety of functional groups close to the sulphoxide have been extensively reviewed [8] and shall not be discussed here, since they bear little resemblance to the alkyl aryl sulphoxides that were produced in the research of the present thesis. The same holds for the introduction of heteroatomic groups to sulphoxides.

There is no doubt concerning the value of chiral sulphoxides as chiral synthons and auxiliaries in organic synthesis, although in the present research the formation of chiral sulphoxides was primarily studied lo serve as a pioneer reaction to determine the conditions for the more difficult enantioselective epoxidalion reactions. Nonetheless, the positive response at international conferences to our enzymatic production of chiral sulphoxides, made clear that especially foreign pharmaceutical industries do have interest in a straight-forward production of the latter.

Justification In the pharmaceutical, agrochemical, and flavour and fragrance industries there is an ever increasing demand for the synthesis of optically pure intermediates. At the moment still expensive (heavy) metal complexes are used as catalysts in the synthesis of these compounds (either direct or via kinetic resolution), causing a burden to the environment. To minimise the waste stream and enantiomeric ballast of these conversions, and to improve upon chemical Chapter 1 yields and stereoselectivity, alternatives are necessary. Biocatalysts are well known for the high degree of selectivity and can offer a favourable alternative if they can be cheaply produced and applied on a large scale. In the present research project catalytic enanlioselective epoxidations and enantioselective sulphoxidations are at stake. Redox enzymes such as peroxidases are potentially suitable biocatalysts for these reactions and meet some of the above mentioned requirements. Some haem peroxidases arc produced by fungi and secreted in the growth medium in large amounts and thus can be easily obtained. These enzymes do not need any (expensive) cosubstrates and use hydrogen peroxide as a clean (cheap) oxidant. A drawback is their lack of operational stability due to oxidative inactivation of the prosthetic group by the substrate

H202. It is known that some of them perform enantioselective oxygen-transfer reactions. Vanadium peroxidases have the advantage that they are not vulnerable with respect to oxidative inactivation. Moreover, they are stable up to high temperatures and in the presence of organic co-solvents up to 40 % [ 15]. Although at the start of this project no oxygen-transfer reactions to organic substrates by vanadium enzymes were reported it was inferred that they might possess such an activity, since vanadium complexes that bear similarity to the prosthetic group of these enzymes, are able to carry out these conversions [16]. Thus at the start of this project these enzymes appeared to be promising, to meet the demands for cheap, clean, natural and stereoselective catalysts. Whether indeed these goals have been attained, will be discussed in chapter 8.

Project Approach To be able to understand the project approach that was aimed to obtain industrially accessible biocatalysts for enantioselective oxygen-transfer, it is necessary to expatiate upon the state of the art at the start of the present research project. As mentioned before CPO has been shown to be an interesting catalyst in the production of (R)-sulphoxides of thioanisole derivatives [4]. However this enzyme did not provide a satisfactory enantioselectivity in the epoxidation of styrene [5]. Horseradish peroxidase had already been shown to catalyse the sulphoxidation of thioanisole to form the (S)-sulphoxides with an e.e. of about 60 %, but this peroxidase did not catalyse the epoxidation of styrene [17]. Via site-directed mutagenesis in a Baculovirus expression system a mutant of HRP was obtained that could produce the (S)-sulphoxide of thioanisole in 97 % e.e. (F41L HRP) and that had some epoxidising activity [18].

10 Introduction

In the research reported here the aim was to use "industrially accessible " biocatalysts, i.e. biocatalysts that can easily and cheaply be isolated and that are relatively stable. Horseradish peroxidase and mutant-HRP do not meet these two criteria. However, a number of fungal peroxidases exists, that are homologous to HRP to a large extent and that are secreted by the fungi into the culture broth, thus offering a favourable alternative: Coprinus cinereus peroxidase (CiP), manganese peroxidase (MnP) and lignin peroxidase (LiP). Moreover these peroxidases are more or less similar in reactivity to HRP. For CiP and MnP at the start of this research project already expression systems existed, that can be considered "industrially acccessible" [19,20]. The research towards the development of an expression system for LiP appeared promising. Further, CiP and mutants of CiP are produced by NOVO Nordisk in Denmark and MnP and mutants of MnP by the group of Prof. Dr. M.H. Gold in Oregon in the USA. Another peroxidase that is industrially accessible is the mammalian peroxidase lactoperoxidase that can be isolated in large amounts from whey which is a side product of cheese making. This enzyme is already applied in the toothpaste Zendium®. Another class of promising enzyme systems arc the vanadium haloperoxidases. The prosthetic group of these enzymes resembles some vanadium-peroxo complexes, that are shown to carry out enantioselective sulphoxidation and epoxidation [16]. In addition for this enzyme an expression system has been developed in Amsterdam, allowing for the production of the enzyme on a gram scale. Research on this enzyme has been carried out predominantly in project 4.

In the present project conditions had to be determined first that were favourable for the production of chiral sulphoxidcs by horseradish peroxidase, Coprinus cinereus peroxidase, and lactoperoxidase. We only disposed of a very small amount of a manganese peroxidase preparation, and for this enzyme only some preliminary results are presented in this thesis. The sulphoxidation reaction would serve as a model reaction to determine or to derive experimental conditions in the catalysis of the more difficult epoxidation that would be investigated subsequently.

To be able to improve upon the e.e. and yield of these conversions the following considerations were at stake : "In the project special attention should be paid to both the accessibility of the active site and the occurrence of non-specific electron-transfer reactions. The performance of the enzymes is strongly influenced by these factors , thereby affecting the

11 Chapter 1 enantioselectivity. To circumvent the problem of competing reactions, modification of the enzymes most probably is necessary to alter the substrate specificity of the enzymes". Initially chemical modifications were planned; in the second phase site-directed mutagenesis was planned to be the "preferred pathway". The presuppositions were the following : 1. In the non-specific electron-transfer reactions almost concomitantly a hydrogen atom is transferred to some site in the protein, most probably a distal histidine" and "In fact modification of the distal hislidine of HRP by diethyl pyrocarbonate (DEPC) allows for formation of Compound I ( the supposed oxygen- transferring enzyme intermediate) but inhibits the formation of Compound II in the oxidation of phenols ( i.e. suppresses the one-electron transfer reactions)" [21]. 2. The incorporation of oxygen in a substrate "proceeds either selectively via oxygen-transfer (concerledly or via a rebound mechanism) or - non-selectively - via electron transfer followed by incorporation, of oxygen from either air or water" . Thus a working hypothesis was formulated assuming "efficient enantioselective oxygen transfer will only occur if the substrate can approach the oxoferryl site (read: the oxygen- transferring species) directly (accessibility of the active site), whereas secondary reactions like longe range electron transfer and protonation of the oxoferryl site should be suppressed. The accessibility is regulated by a combination of steric and electronic factors in combination with the presence of hydrogen bonds. The accessibility can be affected by making a proper choice of the enzyme (crystal structure, screening ) and by modification (chemically and via site-directed mutagenesis [18] J of the amino acids involved in binding the substrate. To suppress the completion of the redox cycle via longe range electron transfer in combination with protonation of the oxoferryl site by the distal histidine, also modification of this and other hislidine residues should be investigated". Therefore DEPC modifications of these enzymes have been attempted and we have requested NOVO-Nordisk to create mutants of Coprinus cinereus peroxidase in which the above mentioned histidine or the adjacent phenylalanine was to be replaced by another amino acid residue. In chapter 8 the results will be evaluated in the light of the objective, justification and project approach.

12 Introduction

Oxidoreductases As mentioned in 1.3, redox enzymes arc potentially attractive biocatalysts in the catalysis of cnantioselective oxygen-transfer. Other biocatalysts will be treated in chapter 2. Redox enzymes are commonly known as oxidoreductases, a class of enzymes, which can be divided into four categories, according to the oxidant being used and the reactions being catalysed : 1. Dehydrogenases 2. Oxidases 3. Oxygenases - Monooxygenascs - Dioxygcnases 4. Peroxidases Dehydrogenases transfer a hydrogen atom from one substrate to the other. Although these oxidoreductases are the most studied ones, with respect to a possible application in organic synthesis, their application has been hampered due to the need for stoichiometric amounts of expensive cosubstrates such as NADH or NADPH in the conversions. Oxidases transfer a hydrogen atom from a substrate to molecular oxygen yielding water or hydrogen peroxide. Oxygenases catalyse the incorporation of oxygen atoms deriving from molecular oxygen into substrate. For monooxygenases only one of the atoms from molecular oxygen is incorporated into the substrate, for dioxygenases both oxygen atoms are incorporated. Again, the application of these enzymes is hampered by the use of expensive cosubstrates (NADH or NADPH) in the conversions catalysed. Moreover these enzymes are difficult to isolate and relatively unstable. Whole cell systems in which these enzymes are present can be used to carry out the required selective oxygen incorporation reactions. Although fairly high enantiosclcctivities and reasonable yields have been obtained with these systems, for most systems known, only R- epoxides were formed as for example in the epoxidation of styrene. Peroxidases and catalases catalyse oxidation reactions in which hydrogen peroxide is the oxidant. The most general peroxidase reaction is the one-electron abstraction from a substrate leading to a substrate radical. No expensive cosubstrates are required and a clean, cheap oxidant is used. Haem peroxidases are in general relatively stable, easy to isolate extracellular enzymes, the same applies to the vanadium peroxidases.

13 Chapter 1

Peroxidases Peroxidases (E 1.11.1.7) are oxidoreductases using hydrogen peroxide or alkyl hydroperoxides as an oxidant. Peroxidases are ubiquitously found in microorganisms, plants and animals [22,23], The nomenclature is either based on their sources, for example horseradish peroxidase, lactoperoxidase and myeloperoxidase or on their substrates , such as chloro, lignin and cytochrome c peroxidase. Most peroxidases studied contain a haem prosthetic group, which is a ferric protoporphyrin IX or a derivative of this if covalently attached to the enzyme. Another important class of peroxidases arc the vanadium haloperoxidases that contain an

2 orthovanadatc (HV04 ) ion as a prosthetic group [24]. Another family of peroxidases, that is not treated in this thesis, is the gluthation peroxidase family containing a selenium cofactor as prosthetic group [25]. Peroxidases fulfil an extremely important role in the protection of life against oxidative damage [26]. One of the requirements of life is to maintain molecules in a reduced state although they are exposed to an oxidising atmosphere. However, partial reduction of oxygen is a wide occurring biological process and thus oxygen intermediates with oxidation states between those of oxygen and water are formed, being the superoxide anion (02~ ), hydroxyl radicals (OH°) and hydrogen peroxide. These highly reactive species are removed efficiently in nature by peroxidases, catalases and superoxide dismutase. In fact peroxidases use the oxidative equivalents thus gained for a variety of interesting oxidation reactions. The different types of reactions catalysed by peroxidases are :

1. One-electron oxidations : 2 AH + H202 -> 2 A° + 2 H20

The formed radicals can undergo all kinds of reactions such as coupling reactions, disproportionations and reactions with molecular oxygen, that shall not be treated here in detail. 2. The disproporlionation of hydrogen peroxide into water and oxygen :

2 H202 -» 2 H20 + 02 this is called catalase activity throughout this thesis, since the older term catalatic activity is often mistaken for catalytic activity by outsiders in the field.

+ 3. Oxidation of halide ions to hypohalous acid : H202 + X + H30 ^ HOX + 2H20

4. Oxygen-transfer reactions : R + H202 —» RO + H20 ; this activity is also called

"peroxygenase" activity.

14 Introduction

Not all these reactions are catalysed by all peroxidases. To be called a "peroxidase" the enzyme must be capable of catalysing at least reaction type 1 or 3.

Haem peroxidases History The first observations of a peroxidase catalysed reaction date from as far back as 1855, when Schönbein detected that samples of animal tissue treated with guaiacum turned blue [27]. Linossier demonstrated in 1898 that an oxidase free preparation of white blood cells from pus catalysed the oxidation of organic compounds present due to the working of an enzyme that he named peroxidase and that is presently known as myeloperoxidase [28]. The first haem peroxidase, horseradish peroxidase was isolated in 1903 [29], Many haem peroxidases have been discovered, isolated and characterised since.

General aspects Most plant and fungal haem peroxidases are small (30-70 kD) monomer proteins [26]. The mass of mamalian peroxidases ranges from 72 kD to 145 kD [30-33]. Of the latter myeloperoxidase is the only dimcr (145 kD) [33]. As mentioned above haem peroxidases contain a ferric protoporphyrin IX or a derivative thereof as their prosthetic group (Fig.9).

HO- OH ^O r~ Figure 9. Ferric protoporphyrin IX ; Lhe prosthetic group of most known haem peroxidases.

In most haem peroxidases the haem iron is coordinated by a histidine residue. In chloroperoxidase from Caldariomyces fumago the iron is however ligated to a thiolatc

15 Chapter 1 .

(cysteine) [34]. This side of the haera, where the iron is ligated to an amino acid residue is called the proximal side. The other side of the haem is called the distal side. At this side the binding of hydrogen peroxide takes place, supported by the so-called distal ligand. For most peroxidases this is a histidine. Again CPO is at odds containing a glutamate at this position [35]. Fig. 10 gives an example of the position of the distal and proximal ligand with respect to the haem group.

A distal ligand

proximal ligand

Figure 10. Position of the distal and proximal ligand with respect to the haem group in the crystal structure of myeloperoxidase.

Most organic one-electron donor substrates are believed to donate their electron at what is called the 5-meso-edge of the haem and are hindered to migrate further towards the iron [36,37]. Substrates to which an oxygen atom is transferred are believed to migrate towards the iron centre [38]. For all known plant and fungal haem peroxidases the haem is not attached to the apoprotein via a covalent linkage and it can be easily extracted. For lactoperoxidase and eosinophil peroxidase the prosthetic group is covalently attached to the apoprotein via two ester linkages between OH-groups on the porphyrin and an aspartate and a glutamate residue[39[. For myeloperoxidase in addition to these two ester linkages, a sulphonium linkage exists between a vinyl moiety of the porphyrin and a methionine residue [40]. There is a striking trend in the ability to oxidise halide ions with respect to the different degree of attachment. Peroxidases with a non-covalently bound protoporphyrin IX are only capable of oxidising iodide [41 ]. LPO and EPO containing two estcrlinkages also oxidise bromide [42,43] and MPO containing three covalent linkages is able to oxidise chloride [44]. It is known that the redox potential of 16 Introduction the oxidising species of peroxidases (the so-called compound I) is influenced by the charge of groups in the neighbourhood of this species [45] and those charges vary with the degree of attachment. The fact that CPO also oxidises iodide, bromide and even chloride (hence its name) is ascribed to its uncommon axial thiolate ligand [46] and its uncommon distal glutamate ligand, that destabilises the oxidising species [47].

Catalytic cycle The traditional haem peroxidase catalytic cycle in presented in scheme 1[48].

Native

Compound II

Scheme 1. The traditional hacm-peroxidasc catalytic cycle. B is the distal ligand that functions as an acid-base catalyst. AH is a substrate. The haem is indicated as —Fe—.

In its resting state the iron in the enzyme is usually present in a high spin ferric state (S=5/2)

7 1 [49]. This reacts with H202 very fast (typically in the order of 10 M'Y ) [50] to yield a highly reactive oxo-ferryl species, known as compound I. This species is formally two oxidation equivalents above the ferric state. A molecule of water is released in this step. It has been shown by Mössbauer [51,52], and EPR [53] that compound I contains an oxoferryl: FclV=0 and a porphyrin 7t radical cation: P+°. In literature this is often abbreviated as FeIV=OP+0. If a suitable reducing substrate (AH) is present, compound I will be reduced via a one-electron transfer to form compound II and a substrate radical A° [26,54]. It is believed

17 Chapter 1

that in this step the distal ligand is concomitantly protonated (BH) [21, 54-56]. A second molecule of substrate reduces compound II back to the native enzyme and a second molecule of water is released [48,54]. Hacm peroxidases also sometimes bypass the formation of compound II and return immediately to the native state. This is believed to be the case in :

1. The catalase activity [26,57]; compound I reacts with H202 yielding H20 and 02. 2. The halogenating activity [41]; compound I forms an adduct with the halogen (which has been described to have special spectral properties [46]) after which hypohalous acid is released. 3. Oxygen-transfer reactions by CPO [58]; compound 1 directly transfers its oxygen to a suitable substrate thereby returning to the native state. This is also known as the oxene mechanism. This mechanism is different for the other haem peroxidases. For these enzymes the mechanism of oxygen-transfer is a variant to this traditional catalytic mechanism and this will be treated in chapter 2. In addition to native enzyme, compound I and compound II, two more valence states are possible for peroxidases. The native enzyme can be reduced by a strong reductant (such as dithionite [59]) to a low spin Fell state. The enzyme in this state is called fcrroperoxidase [60]. In the presence of oxygen ferropcroxidase may coordinate oxygen to form oxyperoxidase [61]. Two resonance structures can be depicted of this state. One structure has the electron mainly on the iron (Fe2+) leaving the oxygen neutral and one structure has the electron delocalised on the oxygen and it can be considered as a superoxide anion coordinated to a Fe III state. This species is also called compound III. Oxyperoxidasc can also be generated by the addition of superoxide

anion [62] or a large excess of H202 to a o o peroxidase [63,64], It is also believed to be Fe III Fe II formed upon reaction of compound II with

H202 [65,66]. Oxyperoxidase is for many peroxidases a catalytic inactive species, leading to the inactivation of the enzyme [67,68], Yet there are reports showing that compound III of myeloperoxidase can return to compound I or native enzyme in the presence of a suitable substrate such as NADH [69], dihydroxyfumarate [70] and cysteamine [71 J. For lignin peroxidase veratrylalcohol [72] and for horseradish peroxidase NADH [73] and indole acetic acid [74]

18 Introduction

have been reported to mediate this event. These reactions are most often a consequence of the peroxidase-oxidase reaction.

Zooming into the mechanism Zooming into the mechanism some details appear to have an important significance with respect to the different reactivities observed for different peroxidases : First, the cleavage of the O-O bond of the hydrogen peroxide is believed to be promoted by the push-pull mechanism [75,76] shown in scheme 2.

His42 Arg3B Arg38 N t* A N© / © H O H OC, H

Fe I

/fc His170

"Compound 0"

His42 Arg38 ,Arg38 /N© X N© H HO. O "H I _ FeC 4 I

% His170 H B© B Scheme 2. The "push-pull" mechanism suggested by Poulos and Kraut for the formation of compound I. In fact one can consider these intermediates as compound 0.

The binding of hydrogen peroxide in the active site has been shown to form first a hydrogen peroxide adduct which has been spectroscopically characterised as a so-called hyperporphyrin

19 Chapter 1

and this species intermediate between native enzyme and compound I is called compound 0

[77]. H202 is transformed into a much better nucleophile upon transfer of its proton to the distal basic group [78]. This nucleophile exerts the so-called push (downward arrow), forming the iron-oxo bond, which can be facilitated if the ligand on the proximal side contains a positive charge, as is the case for a proximal histidine. The charge properties have switched to the other side of the haem and the oxygen bound to the iron exerts a pull (upward arrow), facilitating the cleavage of the oxygen-oxygen bond, assisted by positive charges on the distal ligand (if this is a histidine) and sometimes an adjacent arginine residue, and a negative charge on the proximal ligand if this is also a histidine [22]. The nearby arginine residue not only plays a role in the formation of compound I stabilising the developing negative charge on

the the "OH" " leaving group of H202, but moreover forms a hydrogen bond with the ferryl oxygen atom after the O-O cleavage [79,80].

For CPO, the proximal thiolate facilitates the cleavage of the O-O cleavage even more due to a stronger trans-donating effect in the dz2 orbital causing a stronger pull [57], The presence of a glutamate as distal ligand instead of a histidine creates a more reactive compound I, due to the rigidity of this ligand and its improper positioning to stabilise compound 1 once formed [37].

In cytochrome c peroxidase (CcP) EPR [81] and ENDOR [82] experiments have proved that the radical cation of compound I is delocalised to a nearby aromatic residue (Trp-191). Thus a different compound I species is generated that is often depicted as FeIV=0 R°. This species has a spectrum similar to that of compound II, but contains yet two oxidative equivalents, that are spatially separated. For LPO it is also believed that such a species is formed but the species has never positively been identified [83], In chapter 4 we show that the existence is questionable. Finally, most one-electron oxidations are believed to take place at the 8-meso edge but enols and phenols are believed to directly transfer a proton to the distal ligand in this process and might therefore migrate somewhat further towards the oxo-ferryl [36-38].

Spectral properties The mentioned enzyme intermediates all have been characterised by UV-Vis spectroscopy, thanks to the differences in the electronic distribution over the energy levels of the iron porphyrin ligand system. Most visual properties are a consequence of 71 —»it* transitions in the

20 Introduction

18p electron porphyrin ring [26J. Fig. 11 shows the state diagram of the 18p electron

porphyrin ring assuming D$b symmetry for the porphyrin system.

Common Terminology for bands observed State Svmmetrv Enerav

N Eu Soret, y Eu -24000 cm (420 nm)

n E u -18000 cm Visible | (560 nm) Qoo Eu

G.S.-

Figure 11. Slale diagram of the 18p electron porphyrin ring assuming DAh symmetry for the porphyrin system.

Absorption in the visual region (300-800 nm) is mainly due to transitions from the Aig state to degenerate n* excited states. The lowest energy transitions are identified as a or Qoo band and ß for Qoi, Qo2 etc. A single intense band lies at higher energies and is called the Soret or y- band, with an extinction coefficient of about 105 mM4cm '. At slightly higher energies lies a broader band of weaker intensity, being the third n* state. Absorption in the UV region is mostly due to aromatic residues (tryptophans and tyrosines in particular) of the apoprotein. Charge transfers between the metal and the ring superimpose a series of bands on the 7t —> n* spectrum. For example the band at 700 nm is due to charge transfer from the filled ligand 7t orbitals into the half filled d orbitals of the metal. Figure 12 shows the spectra of native enzyme, compound I, compound II and compound III of horseradish peroxidase. The high spin ferric native enzyme has a single intense Soret band and weak a and ß bands. For compound I the Soret band is about half as intense as for the native enzyme, due to charge transfer from the imidazolate ligand to the iron [84]. Low-spin states as compound II and III have a red-shifted Soret band and distinct a and ß bands.

21 Chapter 1

300 350 400 450 500 600 700 wavelength (nm)

Figure 12. Optical absorption spectra of native HRP, HRP-I, HRP-1I and HRP-HI. - , 10 jiM HRP in potassium phosphate 100 mM (pH 6.5); HRP-I generated by addition of 10 uM hydrogen peroxide; , HRP-II generated by addition of 10 uM ferrocyanide ; ' ' , HRP-HI generated by addition of a large excess hydrogen peroxide i.e. 2.5 mM.

Vanadium peroxidases History

In 1983 Viltcr et al. discovered a fully active non-haem haloperoxidase in the brown alga Ascophyllum nodosum [85,86]. The catalytic activity was found to be dependent on the composition of the buffer solution [85,86]. Subsequently it was discovered [87] that dialysis against EDTA in a citrate /phosphate buffer of pH 3.8 led to an inactivation of the bromoperoxidase and that the enzyme could be reactivated by incubation with vanadium V, whereas other metal ions were ineffective to restore its peroxidase activity. The presence of vanadium as an essential cofactor in these enzymes was positively confirmed by EPR and atomic absorption, Electron Spin Echo (ESE) and Extended X-ray Absorption Fine Structure (EXAFS) [88-94]. Since then many vanadium haloperoxidases have been identified and isolated, not only from other marine sources but also from terrestrial fungi [95].

Characteristics Vanadium peroxidases often exist in the form of clusters of monomers. However, the molecular mass of the monomers usually is between 65-70 kD [96]. These enzymes show a remarkable thermostability and remain active up to 70°C for several minutes or even hours [15,97-100]. The chloroperoxidase from C.inaequalis even withstands temperatures higher than 80°C. Further these enzymes arc stable in the presence of organic solvents that can be mixed with water up to 30 % for several weeks [15, 99,100]. It is known from the crystal structure of chloroperoxidase from C.inaequalis that in the active site the prosthetic group, an

22 Introduction

orthovanadate (HVO4 ") ion is coordinated to a histidinc (His-496) forming a trigonal bipyramide structure (Fig. 13) [14, 101]. H N

O LyS353

O- NHï y^-'-ö. ^ V HN, ^Arg490 HN' H2 ^ ft His \ W 496 Arg360

Figure 13. The prosthetic group of vanadium haloperoxidäses, an orthovanadate (HV04 ) ion coordinated to a histidine (His-496) forming a trigonal bipyramide structure (Hemrika et al. ).

The vanadate is furthermore hydrogen bonded to His-404, Arg-490, Arg-360 and Lys-353. The His-404 seems to play an active role in the mechanism. In addition to the oxidation of halides, haloperoxidäses also possess phosphatase activity [102]. Finally, in this thesis it shown that these enzymes also can catalyse cnantioselective oxygen-transfer [103](see Chapter 5).

Mechanism The mechanism for the oxidation of halides catalysed by vanadium peroxidases as suggested by Hemrika el al. [101] is depicted in figure 14. Upon addition of H2O2 a vanadium peroxocomplex is formed in which the oxygen atoms are bound in a side-on fashion. To form this complex, it is suggested that first a water molecule leaves the active site followed by a hydroxyl ion leading to a complex, in which the vanadium is still surrounded by four oxygen atoms. The His-404 may play an important role in the formation of this complex. One of the side-on bound peroxo oxygens is less electronegative

23 Chapter 1

than the other, rendering the first vulnerable to attack of nucleophiles. When a halide ion X" enters the active site it will attack the oxygen atom which is partly positively charged (S+). A molecule of water may now aid the formation of a hypohalous acid HOX, that will subsequently depart from the active site, leaving the enzyme in its resting state. In this mechanism it is clear in the course of the reaction that the valence state of the vanadium (5+) does not change. H /K H H

o> \ er \ V^ 4\ 0v H H

i,\ i\ il V HOX H H,0 , x 0 4 X rHv / L « 1 \ A °i ] ^oh

Figure 14, The mechanism tor the oxidation of halides by vanadium haloperoxidases as suggested by I Iemrika ci ul [101

Scope of this thesis The goal of this thesis was to study catalytic enantioselective oxygen-transfers in sulphoxidations and epoxidations, by both haem and vanadium peroxidases, with a focus on the mechanism using spectroscopic tools. Chapter 2 is a review on the catalytic enantioselective oxygen-transfer by peroxidases and discusses both the mechanisms as the different techniques used to improve upon cnantioselectivity and yields in these conversions. Chapter 3 describes first the catalytic enantioselective sulphoxidation of thioanisole by HRP, MPO and MnP, concerning techniques used and oddities observed. This functions as an introduction for the catalytic enantioselective sulphoxidation of thioanisole by CiP and LPO and the way this process was optimised.

24 Introduction

Chapter 4 puts a previous suggested mechanism for oxygen-transfer by LPO via a peroxo protein radical into the pillory, by narrating the quest for a protein radical in "compound II" of this enzyme. Moreover it shows that compound II of LPO has the same properties as compound II observed in other peroxidases. Chapter 5 deals with an investigation of oxygen-transfer and other oxidation reactions catalysed by vanadium peroxidases and it is shown that these enzymes catalyse enantiosclective sulphoxidations.

Chapter 6 describes the enantioselective epoxidations of styrene derivatives by CiP and MPO and it is shown for the first time that these peroxidases catalyse the cleavage of a carbon- carbon double bond. Chapter 7 deals with attempts to improve enantioselectivity or yield of enantioselective oxygen-transfers by using chemically modified enzymes. Phenylhydrazine modifications improve the enantioselectivity of epoxidations catalysed by CiP and LPO and lower the enantioselectivity of sulphoxidations catalysed by CiP, LPO and HRP. Chapter 8 gives an evaluation of this research with respect to the objective and justification mentioned in this chapter. Chapter 9 is a summary in English, Dutch and French for outsiders in the field.

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26 Introduction

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27 Chapter 1

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