Iron- and Copper-Based Oxygenation Enzymes: Structure ... · copper-based enzymes, providing a general introduction about the types and functions of the enzymes, followed by a more

Iron- and Copper-Based Oxygenation EnzymesStructure, Function, Models

Bachelor’s Thesis

Ralph KoitzAm Rehgrund 4, 8043 Graz, Austria

Student ID: 0611101

Submitted: Nov. 10th, 2008Supervisor: Univ.-Prof. Dr. Nadia C. Mösch-Zanetti

Contents

Abstract 1

I. Oxygen Activation by Iron and Copper Enzymes 2

1. Oxygen Activation by Iron Enzymes 31.1. Heme-Based Oxygen-Activating Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2. Non-Heme Oxygenation Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2. Copper-Based Enzymes 112.1. Copper in Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.2. Galactose Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.3. Iron and Copper together in one Active Site: Cytochrome C Oxidase . . . . . . . . . . . . . 15

II. Model Compounds for Mononuclear Metalloenzymes 18

3. Presently Available Models for Iron and Copper Active Sites 193.1. Purpose and Significance of Model Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 193.2. Iron Enzymes: Modeling Catechol Dioxygenases . . . . . . . . . . . . . . . . . . . . . . . . . 203.3. Copper Enzymes: Models for Galactose Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . 24

4. Synthesis of Model Complexes 264.1. Research Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264.2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274.3. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

5. Results and Discussion 315.1. Tri- and Tetradentate Ligands for Versatile Coordination Compounds . . . . . . . . . . . . . 315.2. Spectroscopic Evidence for a New Monophenolate Iron(III) Complex? . . . . . . . . . . . . 325.3. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Experimental Procedures for Individual Substances 34RK01 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34RK02 – RK05 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35RK06 – RK13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36RK14 – RK16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37RK17 – RK19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38RK20 – RK21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

References 40

Abstract

Iron and Copper are the two transition metals most commonly found in metal-dependent enzymes, serv-ing a vast number of functions that range from reversible oxygen binding and transport to complex multi-enzyme multi-electron redox cascades. In spite of the fascinating discoveries that have been made overthe last forty years, much remains yet to be learned about the structure and function of iron and copperenzymes.

This thesis is subdivided into two parts. The first part focuses on oxygen activation by iron- andcopper-based enzymes, providing a general introduction about the types and functions of the enzymes,followed by a more thorough examination of specific examples. The first chapter introduces one heme-based oxygen-activating enzyme (cytochrome P450 oxidase), and two non-heme oxygenases, methanemonooxygenases and the catechol dioxygenase family. Special emphasis is put on structure-reactivity re-lationships within the classes of enzymes, whereby the iron cofactor and its role in the enzyme’s reactioncycle is always at the center of attention.

The subsequent chapter expands on copper-based galactose oxidase, as well as an enzyme that relieson both copper and iron, cytochrome c oxidase. Especially remarkable are the closely interrelated func-tions and properties of the two metals, as they work in conjunction to achieve a remarkable four-electronreduction of molecular dioxygen to water in the final step of the respiratory chain.

The second part of the thesis outlines the state of the art on model compounds for the aforementionedenzymes before then going on to provide an account of the research carried out by the author, inves-tigating tri- and tetradentate monophenolate iron(III) and copper(II) complexes, model compounds forcatechol dioxygenases and galactose oxidase. Four new aminophenol chelate ligands and a novel tripodaliron(III) complex are reported.

The practical research that led to the compounds described in the second part was carried out betweenSeptember 1st and 19th, 2008 under the supervision of Prof. Dr. Nadia C. Mösch-Zanetti and Mag. MartinaJudmaier at the Institute of Chemistry: Inorganic Chemistry at the University of Graz. Without theirgenerous support in all matters, as well as the kind assistance extended by all members of the researchgroup, the writing of this thesis would not have been possible.

For that I wholeheartedly thank them.

1

Oxygen Activation by Iron andCopper Enzymes

PART I

1Oxygen Activation by Iron Enzymes

Iron is the most common transition metal in biological systems, found in a great variety of metalloen-zymes as well as non-enzymatic metalloproteins. Iron enzymes can be further subdivided into two fun-damentally different groups.1 In the first, the so-called heme-based iron enzymes, the iron atom is coordi-nated by a porphyrin-ligand, forming a heme-group. Non-heme iron enzymes, on the other hand, containone or more iron atoms coordinated by various other ligands, predominantly with oxygen, nitrogen andsulfur atoms.

Aside from O2-transport and storage by proteins such as hemoglobin and myoglobin (heme-systems)and hemerythrin (non-heme), numerous iron enzymes are responsible for the activation and utilizationof dioxygen in oxidation and oxygenation reactions.

After a general introduction and a cursory overview of heme-based oxygenation enzymes, this chap-ter focuses on mononuclear non-heme oxygenases, especially on the structure and function of MethaneMonooxygenase and Catechol Dioxygenases.

1.1. Heme-Based Oxygen-Activating Enzymes

The common element in all heme-dependent enzymes and metalloproteins is an iron-porphyrin complex,referred to as the heme group. An iron(II) or iron(III) ion is coordinated in the center of the fully conjugatedplanar ring system of protoporphyrin IX as shown in fig. 1.1. Depending on the exact nature of the protein,additional amino acid residues or other molecules constitute the two remaining axial ligands. Table 1.1lists the axial ligands of different heme proteins, along with the oxidation state of the metal ion in theresting state and the function of the protein. Aside from heme proper, other tetrapyrrole ligands such aschlorin and siroheme also occur as the iron-containing moieties in certain active sites.2

Heme-based enzymes are crucial for electron transport and accumulation, conversion of oxygen-con-taining intermediates, as well as complex redox processes. A large number of all known heme enzymes

N

N N

N

HOOC COOH

Fe

Figure 1.1.: The Heme Group (Fe + Protoporphyrin IX)

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Bachelor’s Thesis Ralph Koitz

Protein Function Metal Axial Ligand(s)

Hemoglobin O2 transport Fe(II) His

Cytochromes Electron transport Fe(II) His (+Met)

Cytochrome P-450 Monooxygenation Fe(III) Cys-

Peroxidases Peroxide reduction Fe(III) His

Cytochrome c Oxidase Dioxygen reduction Fe(III) His

Table 1.1.: Different Heme Proteins: Axial Ligands and Oxidation States2

utilize oxygen in the form of molecular dioxygen or so-called ‘active oxygen intermediates’ such as per-oxides. Among them are peroxidases, heme oxygenases, catalases, and nitric oxide synthetases, making theheme group an extraordinarily flexible cofactor. Its versatility hinges on the sequence and structure of theprotein backbone as well as on the porphyrin ligand and its vacant axial binding sites.

Most of the total iron in mammals is used for the synthesis of heme proteins, mainly hemoglobin, myo-globin, and cytochromes. The assembly of the heme group is carried out by ferrochelatase, which catalyzesthe insertion of Fe(II) into the protoporphyrin macrocycle. The latter is synthesized in a complex path-way, consisting of four general steps: synthesis of the pyrrole moiety, condensation into a tetrapyrrole,modification of the side chains, and final oxidation of protoporphyrinogen IX to protoporphyrin IX.3

1.1.1. Cytochrome P450: A heme-based monooxygenase

The class of cytochromes P450, abbreviated CYP, is an important member of the superfamily of oxyge-nases. These enzymes, named P450 due to a characteristic absorbance of the CO-complex at 450 nm, areheme-based monoxygenases. They utilize molecular oxygen in conjunction with a reducing agent suchas NADH to oxygenate inert substrates such as hydrocarbons and olefins.2

CYPs have two general functions: Firstly to metabolize xenobiotics in order to prepare them for excre-tion, thus primarily fulfilling a detoxification function. Secondly, they play a role in the biosynthesis ofsignaling molecules used for control of development and homeostasis. For instance, they are involved inthe synthesis of steroid hormones and the metabolism of certain vitamins in mammals.4 So far more than8000 different types of CYPs have been reported∗ and the number is steadily growing.

In order to be able to detoxify as efficiently as possible, CYPs have very broad substrate specifici-ties, metabolizing a vast array of (predominantly lipophilic) xenobiotic as well as endogenous substrates.The reactions catalyzed by P450-enzymes include hydroxylations, epoxidation, peroxygenation, deamination,desulfuration, and dehalogenation.5 Acting on xenobiotics, these reactions aim at increasing the solubility ofthe substrates, thus facilitating their excretion. In some cases however, e.g. with benzene and benzo[a]-pyrene, these reactions lead to cancerogenic products, ultimately resulting in an increase of toxic sub-stances rather than detoxification.

Structure of Cytochrome P450

Figure 1.2 shows the three-dimensional structure of the folded core of cytochrome P450. Even though only20% of the protein sequence is identical across the family, the fold topology of the core remains highlyconserved. The structure is comprised of a bundle of three parallel helices D,L,I, and one antiparallelhelix E. The heme group is located between helices I and L. A conserved cysteine residue, present in itsdeprotonated form as a thiolate ligand coordinates to the heme-Fe atom perpendicular to the porphyrinplane. This proximal Cys- generally forms two additional hydrogen bonds with amides of the protein∗http://drnelson.utmem.edu/CytochromeP450.html

4


Figure 1.2.: The three-dimensional structure of cytochrome P4504

backbone. Slight variations of the P450 core structure have significant effects on the chemical propertiesof the enzyme, such as redox potential and catalytic activity.

In spite of the highly conserved structure, variations within the P450 family allow for a broad rangeof substrate specificities, and regio- and stereoselectivity. Substrate recognition is accomplished by sixSubstrate Recognition Sites (SRS), as shown in fig. 1.2. The substrate specificity of the P450 enzyme dependson the sequence and structure of these SRS. The SRS change conformation upon substrate binding, thusmaking possible enzyme-substrate interaction and the catalytic reaction.4

CYPs are further subdivided into four classes, depending on the type of electron shuttle used to trans-port reducing equivalents to the metal center. Class I CYPs receive electrons from a specific ferredoxin,which in turn receives electrons from a NAD(P)H dependent reductase. Class II CYPs require a NADPH-cytochrome-P450 reductase, while Class III does not require an electron donor at all. Class IV CYPsreceive electrons directly from NADH.6

The CYP Catalytic Cycle

The reaction mechanism, common to all P450 enzymes, was already proposed in 1968 and has since beenconfirmed and complemented by ample experimental and theoretical evidence. Methods such as X-raycrystallography, advanced spectroscopic techniques, cryogenic isolation of intermediates and quantumchemical calculations have led to a more or less clear picture of the 8-step CYP catalytic cycle, depicted infigure 1.3.

The cycle starts with the low-spin Fe(III) center in octahedral coordination, the plane of the porphyrinring constitutes the four equatorial ligands, while the proximal cysteinate and a molecule of water occupythe axial positions. Hydrophobic interaction with the protein chains results in binding of the substrate(RH) in close proximity to the distal H2O molecule (1). Substrate binding results in a change of spin stateform low spin to high spin, with a concomitant change in reduction potential from roughly -300 mV toabout 100 mV more positive. The relatively low reduction potential of Fe(III) in the resting state of theenzymes causes the iron center to remain in its ferric oxidation state.

As the substrate binds to the protein, the water ligand is perturbed and dissociates from the coordina-tion sphere. A structural rearrangement occurs and the iron center adopts a highly distorted out-of-plane

5


Figure 1.3.: Catalytic Cycle of Cytochrome P4504

geometry (2). CYPs show a very pronounced flexibility in their protein fold and structural changes onthe order of 10 angstroms can be observed. This malleability of the active site is evidence of an induced-fitbinding as opposed to a classical lock-and-key principle.

The next step consists of the reduction of Fe(III) to Fe(II) by FADH2, which then, due to its distortedgeometry and S=2 spin facilitates binding of a molecule of triplet oxygen 3O2 (3).

The ferrous center once again reverts to a low spin diamagnetic species, forming a dioxygen complex(4). As yet, it is unclear whether this species is actually a Fe(II)-O2 or a Fe(III)-O2- complex. However,recent evidence suggests only partial electron transfer from the iron center to the coordinated dioxygen.Oxygen is coordinated end-on with an Fe-O-O angle of 142◦, as determined by X-ray crystallography. Thestructure of this dioxygen adduct strongly resembles its analogues found in myoglobin, hemoglobin andvarious other oxygen-binding heme-enzymes.

Subsequent single-electron reduction yields a peroxo-complex (5a), which absorbs a proton to yieldthe hydroperoxo species (5b).

As the hydroperoxo-intermediate is protonated a second time, the O-O bond is cleaved heterolytically,resulting in a free H2O molecule and a highly reactive Fe(IV)-oxo π-cation radical, referred to as Com-pound I (6). The protoporphyrin moiety in this case functions as a temporary electron donor, whichresults in the formation of a radical cation delocalized on the ring. While this is clearly the key step inthe reaction cycle, characterization of this intermediate has been extremely difficult due to its unstablenature. The compound is more easily accessible via the so-called oxidase-shunt, which bypasses the restof the cycle, going directly from species 2 to 6 using peroxyacids as external oxidizing agents. This hasallowed spectroscopic identification of Compound I.

The catalytic cycle concludes with a two-step oxygenation of the substrate associated to the proteinshell. The free radical is transferred from the π-system to the oxygen atom, which in turn abstracts a hy-drogen radical from the substrate. The hydroxy ligand rebounds as a radical back to the substrate radical,yielding a hydroxylated substrate and leaving behind an electron on the iron center, thereby reducing itto Fe(III) (7). The substrate dissociates and is replaced by a weakly associated water molecule.2,4

6


1.2. Non-Heme Oxygenation Enzymes

Having introduced heme-based iron enzymes using the example of cytochrome P450, this section nowfocuses on non-heme iron oxygenases.

After some general remarks, the structure-reactivity relationships within this vast and highly diversegroup of enzymes will be introduced with two prominent examples, methane monooxygenase and the groupof catechol dioxygenases.

1.2.1. Methane Monooxygenase

A very prominent and fascinating iron-oxo enzyme is methane monooxygenase (MMO)7. MMO is capableof catalyzing a remarkable chemical transformation, the oxidation of methane to methanol at ambienttemperature (equation 1.1). It evolved in methanotrophic bacteria that are dependent on CH4 as both acarbon and an energy source.

CH4 + O2 + NADH + H+ → CH3OH + H2O + NAD+ (1.1)

Two types of MMO are found in methanotrophs: Membrane-bound particulate MMO (pMMO) thatuses multiple copper clusters as redox-active cofactors, and soluble MMO (sMMO) that is produced bysome methanotrophs under conditions of low copper availability. While pMMO is the more commonenzyme, it is more difficult to isolate and purify and therefore less well-characterized.

Soluble MMO, on the other hand, has attracted a lot of attention and has been studied intensively. Theenzyme can not only metabolize CH4, but also various branched and unbranched hydrocarbons with alength of up to 8 carbon atoms, as well as aromatic, heterocyclic and chlorinated compounds. Structurally,sMMO actually consists of three distinct enzymes that work together in order to achieve substrate oxida-tion: The oxidation of CH4 occurs at the active site of the hydroxylase protein (MMOH), which contains adiiron center, bridged by two carboxylate ligands. The electrons required for the reduction of molecularoxygen are provided by the reductase protein (MMOR) through oxidation of NADH. A third, regulatoryprotein (MMOB) is additionally required for catalysis.

While all three enzymes are required for the catalytic cycle of native MMO, only MMOH is responsiblefor dioxygen activation and methane oxidation. MMOH is itself a dimer of three subunits, (αβγ)2, whereeach of the α subunits contains a carboxylate- and hydroxo-bridged diiron center. Figure 1.4 shows thethree-dimensional structure of the active site of MMOH. The two iron centers are coordinated by fourglutamate residues and two histidines contributed from the protein backbone, as well as one water andtwo bridging hydroxo ligands from the solvent.

Figure 1.4.: Active Site of MMOH

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The MMOH Catalytic Cycle

The catalytic cycle of MMOH initiates with the binding of O2 to the reduced diiron (II) center. The ironcenters are subsequently oxidized to +III and a µ-peroxo species forms; the peroxide then decays to twoµ-oxo ligands bridging two Fe(IV) centers. The substrate is then putatively hydroxylated via a bound-radical mechanism: A hydrogen atom is abstracted from the substrate to an oxygen atom, which subse-quently rebounds to the substrate radical. The cycle concludes by reduction of the iron centers by NADH.

1.2.2. Catechol Dioxygenases

Catechol dioxygenases are a class of enzymes utilized by bacteria for the degradation of aromatic com-pounds. The enzymes employ an iron(II) or iron(III) cofactor in order to catalyze the reaction of di-hydroxy aryl compounds (compounds containing a catechol moiety) with molecular oxygen to give acyclicaliphatic products. Both atoms of the dioxygen molecule are integrated into the product, thus avoidingthe need for additional reduction equivalents coming from NAD(P)H or other cofactors. Depending onthe position of the cleavage site in the aromatic ring, catechol dioxygenases are categorized in two dif-ferent groups: Intradiol Dioxygenases catalyze the cleavage of the C-C bond ‘between’ the two hydroxylgroups, while Extradiol Dioxygenases cleave the molecule at a site adjacent to the diol.8 Fig. 1.5 comparesthe two methods of aryl cleavage.

OH

OH

COOH

COOH

COOHCHO

OH

intradiol

cleavage

extradiol cleavage

Figure 1.5.: Intra- and Extradiol Cleavage by Catechol Dioxygenases

Since they show no significant sequence homology, it has been determined that intra- and extradiolcatechol dioxygenases are two evolutionarily separate families. Additionally, sequence evidence suggeststhat all intradiol dioxygenases share a single common ancestor from which they have diverged. Extradioldioxygenases, on the other hand, belong to three distinct families that evolved independently from eachother. Type I, II and III extradiol dioxygenases differ from each other with regard to active site residuesand structural fold.

With regard to the iron cofactor, there is one major difference between intradiol and extradiol dioxy-genases. While intradiol dioxygenases employ a mononuclear iron(III) center, ligated by two tyrosine andtwo histidine ligands, extradiol dioxygenases require an iron(II) center, coordinated by two histidine andone glutamic acid residue.9

Extradiol dioxygenases are more versatile than their intradiol counterparts.10 While intradiol-cleavingenzymes require substrates with two vicinal hydroxy-groups, extradiol dioxygenases are much broaderin their substrate specificities. They are able to cleave non-catecholic compounds such as hydroquinone,salicylate, and 2-aminophenol, as well as halogenated substrates. Also, intradiol enzymes are unableto oxygenate substrates with strongly electron-withdrawing substrates (e.g. nitrocatechol), whereas ex-tradiol enzymes can cleave them, albeit at a slower rate. This difference in the nature of the substratessuggests that there are significant differences in the catalytic mechanisms of the two groups of enzymes.Figure 1.6 lists a number of substrates for catechol dioxygenases as they occur in aromatic degradationpathways.

8


OH

OH

(a)Hydro-quinone

OH

OH

COO-

(b) Protocate-chuate

OH

OH

OH

(c) 1,2,4-trihydroxybenzene

OH

OH

(d) Catechol

COO-

OH

(e) Salicylate

NH2

OH

(f) 2-Aminophenol

Figure 1.6.: Example Substrates for Intradiol Dioxygenases (b-d) and Extradiol Dioxygenases (a-f)

Extradiol Catechol Dioxygenases

Crystal structures of extradiol catechol dioxygenases from numerous species are now available and offerinsights into the selectivity and reactivity of these enzymes. Although the structural folds differentiatethem into three families, all extradiol dioxygenases share the same three iron-binding residues in theactive site.

(a) as-isolated (1KW3.pdb) (b) substrate bound (1KW6.pdb) (c) substrate + NO bound (1KW8.pdb)

Figure 1.7.: Crystallographic Structures of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC); Source: Protein Data Bank (PDB)

The active site of extradiol catechol dioxygenases contains an iron(II), or in some cases manganese(II)center, coordinated in a square pyramidal geometry by two His and one carboxylate residue, in a so-called 2-His-1-carboxylate facial triad.8 This metal-binding motif is common among all families of extradioldioxygenases and is also found in numerous other non-heme iron(II) enzymes. Since the three amino acidresidues all bind to one face of the coordination sphere, there is ample room for the binding of substratesor other ligands on the opposite face. This accounts for the wide range of possible reactions catalyzedby enzymes of this type and has led some authors to consider the 2-His-1-carboxylate facial triad thecounterpart of the heme cofactor.10

Figure 1.7 shows the three-dimensional structure of a typical dioxygenase active site: (a) as-isolated,(b) with a bound substrate, and (c) with substrate and a molecule of NO bound.

Upon binding of the substrate, the two water molecules are displaced and the active site assumes asquare pyramidal geometry, ready to bind an oxygen molecule (or a molecule of NO as shown in thecrystal structure). One oxygen atom of the bidentate catecholate occupies the site previously vacant inthe native enzyme, while the other one coordinates to the site trans to His209. The coordination sitetrans to the carboxylate residue remains vacant, providing a free site to bind oxygen. The two catecholate

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oxygen residues are bound asymmetrically to the iron center, with differences of 0.2-0.4 angstroms inbond lengths. For this reason, it is very likely that the catechol is bound to the Fe center as a monoanion.11

The catalytic cycle of extradiol dioxygenation starts with the activation of dioxygen at the iron center.As the molecule binds to the Fe(II) atom, an electron is transferred, which results in the formation of anFe(III)-superoxide species. Subsequently, the first C-O bond between the substrate and the superoxideis formed, resulting in heterolytic cleavage of the O-O bond. After a rearrangement resulting in a 7-membered ring, the second C-O bond is formed and finally the cleaved product dissociates from theactive site.9

Intradiol Catechol Dioxygenases

In contrast to their extradiol counterparts, intradiol dioxygenases employ an iron(III) cofactor. The metalcenter is coordinated in a trigonal bipyramidal geometry by two tyrosine residues (one axial and oneequatorial), two histidines and one hydroxide. The +III charge on the Fe center and three anionic ligandsresult in a charge-neutral complex. Due to a ligand-to-metal charge transfer by the tyrosinate ligands, theenzymes are red-brown in color.12

As the substrate binds to the active site, the catechol moiety chelates the iron center while donating oneof its protons to the hydroxide and the other one to the axial tyrosinate. Analogous to catechol bindingin extradiol-cleaving enzymes, the two Fe-Ocat bond lengths are not equal. Since one Ocat binds trans toHis460 and the other one trans to Tyr408, they are subject to unequally strong trans effect influences. Thisasymmetric binding is thought be a key feature of the catalytic mechanism.8

Ample experimental evidence suggests a catalytic mechanism that is very different from the extra-diol cleavage mechanism mentioned above. Intradiol dioxygenation works by substrate-activation ratherthan oxygen activation. As the catechol binds to the iron cofactor it is activated and prepared for directinteraction with moleculare dioxygen. The first step of the catalytic cycle consists of the aforementionedchelation of the Fe(III) center by the catecholate, concommitant with deprotonation. This results in atransient semiquinonate radical species that is prone to attack by dioxygen, resulting in an alkylperox-oiron(III) intermediate. Following a Criegee-type rearrangement, a seven-membered cyclic anhydrideforms which is oxygenated again, finally resulting in the intradiol-cleaved acyclic product.

A number of model compounds that mimic the structure and/or function of catechol dioxygenaseshave been synthesized and will be discussed in further detail in part II of this thesis.

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2Copper-Based Enzymes

Primarily, this chapter aims to provide the necessary theoretical and mechanistic background for thediscussion of galactose oxidase models in section 3.3. Since iron and copper have such similar functionsin oxygen turnover, an enzyme that utilizes both copper and iron cofactors, cytochrome c oxidase, willalso be discussed.

2.1. Copper in Biological Systems

Next to iron and zinc, copper is the thirdmost common essential transition element in humans. Copper-dependent proteins, both with enzymatic and non-enzymatic functions, cover a broad array of biochem-ical functions that often closely resemble those of iron-based equivalents. Table 2.1 lists a number offunctions covered by both copper and iron in very similar processes. A number of enzymes, such ascytochrome c oxidase, also employ a combination of iron and copper cofactors.

However, despite some superficial commonalities regarding their functions, copper and iron have verydifferent characteristics in biological environments. While the most common oxidation states of Fe are+II and +III, copper generally alternates between +I and +II, having a higher redox potential than iron.This gives copper-based enzymes the ability to oxidize Fe(II) to Fe(III) as exemplified by the protein ceru-loplasmin. Also, there is no copper-binding equivalent to the heme group; instead, copper is generallycoordinated by nitrogen and sulfur atoms in protein residues such as histidine, cysteine, and methionine.

Structurally, copper centers are classified in three different groups. Type I, so called ‘blue’ copper cen-ters, contain a mononuclear Cu center coordinated by two histidines, one cysteinate, and one methionineligand in a highly distorted tetrahedral geometry. Type II copper enzymes, on the other hand, have aplanar square geometry with three histidines and one other ligand (H2O). Due to Jahn-Teller stretch ofthe Cu coordination sphere, additional axial ligands are weakly associated. Type III copper proteins (e.g.hemocyanin) are Cu-X-Cu dimers, the two copper centers bridged e.g. by O2. Additional coordinationsites are occupied by histidines.2

Function Fe-based protein Cu-based protein

O2 transport Hemoglobin, Hemerythrin Hemocyanin

Oxygenation Cyt P450, MMO, Catechol Dioxygenases Tyrosinase

Oxidase-activity Peroxidases Amine Oxidases, Laccase

Electron Transport Cytochromes blue Cu proteins (Azurin)

Table 2.1.: Copper Equivalents to Iron-Dependent proteins2

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(a) Folded GAO Protein14 (b) Active Site15

Figure 2.1.: Structures of Galactose Oxidase

2.2. Galactose Oxidase

Galactose Oxidase (GAO) is an extracellular fungal enzyme that catalyzes the oxidation of primary alco-hols to aldehydes with molecular dioxygen. As is evident from equation 2.1, the overall reaction catalyzedby GAO results in the reduction of O2 to H2O2, the electrons being withdrawn from the alcohol substrate.While GAO only utilizes a single copper ion as a cofactor, it nevertheless achieves a two-electron oxida-tion in a single step. This is made possible by an unusual free tyrosyl radical in the vicinity of the metalcenter. As yet, the purpose of GAO is not entirely evident. It has been suggested that it provides H2O2necessary as an oxidant for certain fungal peroxidases or is used as an antibiotic defense.13

RCH2OH + O2 → RCHO + H2O2 (2.1)

After an overview of the protein and active-site structure of GAO, this section goes on to describe thepeculiar mechanism of metal/radical based oxidation.

2.2.1. Structure of Galactose Oxidase

Fig. 2.1(a) shows the folded protein structure of GAO. The enzyme is comprised of three different sub-units. Starting from the N-terminus, the first 155 residues comprise a globular domain that includes acarbohydrate binding site, possibly responsible for targeting the enzyme. The actual catalytic domain ismade up of the subsequent 397 residues, forming a β-propeller motif. The very center of the propellercontains the active site with its single copper atom. The last subunit (C-terminus) caps the propeller andprovides one of the metal-binding histidines in the active site.14

The metal-binding site of GAO involves four protein residues from distant regions of the backbonethat only come together in the folded protein. As shown in fig. 2.1(b), the copper atom is coordinatedin a distorted square pyramid, where Tyr495 occupies the axial position and His581, His496, Tyr272 anda solvent-derived ligand or the substrate bind equatorially. GAO is the only metalloenzyme with a bis-tyrosinate coordinated center and this fact plays a key role in the catalytic mechanism. An additionaltryptophane residue in the vicinity is π-stacked with Tyr272, providing controlled access to the active siteand shielding from solvent molecules.

The Cu center can assume +I and +II oxidation states, thus logically allowing only the transfer of oneelectron at a time. Therefore, a second redox-active cofactor is required for a two-electron reduction tooccur in one step. Tyr272 has been determined to function as a radical species, thus providing the secondelectron. Thus three different oxidation states are possible for the active site complex. The reduced form

12


consists of Cu(I) and tyrosinate, making the center EPR-silent and colorless. The semi-oxidized form ofthe complex consists of a Cu(II) center and tyrosinate, but is not catalytically active. This form showsspectroscopic features typical of a mononuclear Cu(II) complex. The actual reactive intermediate of theGAO active site complex is the fully oxidized form Cu(II)/tyrosyl. The radical and the unpaired electronshow strong antiferromagnetic coupling, resulting in a diamagnetic species.13

A very peculiar feature of the GAO active site is a covalent connection of Tyr272 and Cys228 via athioether bond. This o-substitution of the tyrosine is thought to lower its redox potential, making theradical species more accessible and stabilizing it. This aryl-S-alkyl cross-link is the result of a post-translational modification, a self-processed reaction requiring only copper and dioxygen. The enzymeis synthesized in a pro-sequence form and, under conditions of low copper availability remains this way.Only after addition of Cu(II) and aerobic incubation, the modified thioether-link actually forms with con-comitant pro-sequence cleavage.16

The second tyrosine residue in the active site, Tyr495, most likely functions as a general base duringcatalysis. Experiments that replaced the Tyr residue in question by Phe resulted in a more than 1000-folddecrease in catalytic activity, lending support to this hypothesis. The remaining two histidine ligandsappear not to be involved in the catalytic function and do not undergo significant geometrical rearrange-ments during the reaction cycle.14

2.2.2. Mechanism of Galactose Oxidase: Free Radical Catalysis

As is the case also with many iron-based oxygen-activating enzymes, catalysis by GAO proceeds via aradical mechanism. In this case, however, the radical is located on the protein itself, rather than on thecofactor or the oxygen atom.

Fig. 2.2 depicts the four-step reaction cycle of GAO. The elucidation of this (putative) mechanism reliesprimarily on a combination of indirect evidence, since no intermediates have yet been isolated and re-solved. The cycle itself is a rather simple process consisting of only four steps during which, superficiallyspeaking, two protons and two electrons are transferred from the alcohol substrate to dioxygen. Thereaction proceeds as a sequence of steps of proton transfer, single electron transfer, and hydrogen atomtransfer.14

Figure 2.2.: The four-step reaction mechanism of Galactose Oxidase13

13


Part I: Substrate Oxidation

The catalytic cycle of GAO starts by a coordination of the substrate ROH to the fully oxidized (Cu(II),Tyr·) cofactor. A number of protein residues in the vicinity assist in holding the rest of the substratethat is not being oxidized in place. Due to the rather open and easily accessible active site GAO showsbroad substrate specificity and a low affinity constant for the formation of this initial complex. The stericsituation inside the active site, however, strictly controls the orientation of the substrate and allows forstereoselectivity.

Coordination of the alcohol to the metal center lowers its pKa to a significantly more acidic value,allowing for facile proton abstraction. The Tyr495 residue, previously coordinated to the metal center, isprotonated by the alcohol in a fast (nanosecond) proton transfer reaction. The alkoxide ion has a strongeraffinity to the copper center, and thus strengthens the bond to the cofactor. Additionally the bond energyof the substrate C-H bond is lowered by about 40 kJ/mol, preparing the substrate for the next reactionstep.

In the second step, a hydrogen radical is transferred from the alkoxide to the Cys-Tyr radical. Thistransfers the radical character to the now three-valent substrate carbon atom, forming a transient ketylradical. C-H bond cleavage occurs via a five-membered cyclic transition state and is thought to rely atleast partly on hydrogen atom tunneling. Under conditions of high dioxygen availability this step is therate-determining step of the reaction sequence, with an estimated activation energy for hydrogen atomabstraction of 56.9 kJ/mol.

The ketyl radical subsequently reduces the metal center to Cu(I), leading to the aldehyde product. Sincethe latter has a much lower affinity to the cofactor, it is released and replaced by dioxygen, which initiatesthe second half of the reaction cycle, regeneration of the oxidized cofactor.13,14

Part II: Reoxidation of the Cofactor

Even though dioxygen reduction and the formation of hydrogen peroxide are believed to be the ulti-mately ‘useful’ part of GAO function, much less is known about this second half-reaction of enzymeaction. As the aldehyde product is formed, the copper-tyrosine cofactor is left in its fully reduced stateand has to be regenerated. In vitro this reoxidation occurs via O2 reaction, but is also achievable byalternative oxidants such as hexacyanoferrate(III).

The reduced cofactor is oxygenated by the coordination of molecular dioxygen, which subsequentlyundergoes a two-electron reduction. The metal center is reoxidized to Cu(II) and the Cys-Tyr· radical isformed again. The peroxide anion is protonated by the two protons previously abstracted from the sub-strate (as confirmed by isotope labelling) and hydrogen peroxide dissociates from the active site, leavingthe cofactor fully reduced and ready for another turnover.

Since the occurence of partially reduced reactive oxygen intermediates (superoxide, in this case) isextremely toxic and detrimental to enzyme performance, the protein has to be optimized to avoid pre-mature release of partially reduced oxygen. GAO is particularly selective and the amount of superoxideresulting from incomplete reduction is estimated to be less than 1 in 2000. Also, further reduction ofalready formed peroxide does not occur either, making GAO an enzyme extremely well-adapted to its(presumed) function of H2O2 production.14

14


2.3. Iron and Copper together in one Active Site: Cytochrome COxidase

Cytochrome c oxidase, abbreviated COX, is the terminal enzyme of the respiratory chain. It catalyzesthe reduction of molecular oxygen to water, thereby utilizing four reduction equivalents from reducedcytochrome c molecules. At the same time, four protons are translocated across the mitochondrial mem-brane, creating an electrochemical gradient that is utilized by ATPase to store energy in the form of ATP.Equation 2.2 shows the overall coupled reaction; the protons on opposite sites of the membrane are des-ignated by subscripts.17

4 CytC2+ + O2 + (4+n) H+in → 4 CytC3+ + 2 H2O + nH+out (2.2)

The value of n varies between 1 and 4, depending on how many protons are translocated for each elec-tron. Four of the protons result from the fact, that cytochrome c donates its H+/e- reducing equivalentson the outside of the membrane, while the water-forming protons are taken from the inside. Additionalproton translocation occurs through the pumping of vectorial protons from the inside to the outside.

The functional domain of COX contains a total of five redox-active metal centers: two iron and threecopper atoms. Iron is present in two modified heme groups residing in subunit I that are not covalentlylinked to the protein. The three copper ions, referred to as CuA (which is actually a dimer of two Cys-bridged copper atoms in very close proximity) and CuB are located in different subunits. CuB is presentin subunit I, while CuA is found in subunit II. Additionally, one Zn2+, one Mg2+ and one (putative) Ca2+

ions are found in the protein. Those, however, do not serve catalytic functions.

Heme-Iron in Cytochrome c Oxidase

COX contains two heme-iron groups with different ligands and functions, but in close proximity to eachother. Figure 2.3 shows the active center of subunit I of COX. Heme a3 forms a binuclear center with CuB(Cu-Fe distance 4.9 angstroms), at a distance of about 13 angstroms to heme a. In the fully oxidized formof the enzyme, a peroxo-group is found bridging the two metal centers of the a3 complex.

Heme a contains a low-spin Fe(III) center, coordinated axially by two histidine residues. Heme a func-tions as an electron-carrying cofactor, receiving an electron from CuA (located in subunit I, 11.7 angstromsaway), which in turn is acquired from the oxidation of cytochrome c. The electron is then rapidly trans-ferred to the a3 complex. Heme a has a high redox potential, meaning that the reduction of the Fe(III) ionis thermodynamically favored, thereby allowing for a ‘down hill’ electron transfer cascade between themetal centers.

The iron center in heme a3 is present in a high-spin configuration, coordinated axially by a single his-tidine residue. In the fully oxidized state, the a3 complex is comprised of a Fe(III)-µO22--Cu(II) moiety,such that the iron center assumes a roughly octahedral geometry next to a distorted square-planar cop-per complex. The two metal centers are antiferromagnetically coupled, meaning that the 5/2 spin of theiron center and the 1/2 spin of the copper center compensate each other and result in a macroscopicallydiamagnetic species. The oxidized a3 complex is therefore EPR silent. The reduced complex, on the otherhand, contains a Cu(I) center with a fully saturated d10 configuration and a high-spin Fe(II) ion. The un-paired electrons favor the binding of paramagnetic 3O2. The two heme-groups a and a3 are spaced only4 angstroms apart at the point of closest approach, and the axial His378 of heme a is only one residueremoved from His376 of heme a3. This spatial and covalent proximity are ideal for the electron trans-port from one heme to the other, the next step in the electron transfer cascade originating at Cyt c andeventually reaching O2.2,18

15


Figure 2.3.: Active Site Structure of Bovine Heart COX, subunit II19

Figure 2.4.: Oxygen Reduction Mechanism in COX20

Mechanism of COX-catalyzed O2 Reduction

A host of different experimental methods has been utilized in the elucidation of the mechanism of cy-tochrome c oxidase. Predominantly spectroscopic methods have shed light on transient species that occuronly for fractions of a second in the catalytic cycle.

COX can bind and reduce up to 250 molecules of O2 per second, using dioxygen as terminal electronacceptor and thermodynamic sink. At the same time, the enzyme must ensure that partially reducedoxygen species such as superoxide (O2-) and peroxide (O22-) are immediately converted further and notreleased. All this is accomplished with a minimum of waste of electrochemical potential, at room tem-perature and with very high efficiency.

As is the case with many heme-based oxygen-activating enzymes, the catalytic cycle of COX starts withthe formation of an O2 adduct to the fully reduced heme a3 (referred to as Compound A). This step has ahalf-life of approximately 10 µs, forming a product similar to oxygenated hemoglobin.

Depending on the availability of reducing electrons on heme a, the cycle can proceed in two ways. Ifan electron can be rapidly transferred from heme a to the bound oxygen molecule, a peroxidic species PRis formed, containing an Fe(IV) (ferryl) species and Cu(II). At the same time, the electron-donating heme

16


a is oxidized. This intermediate decays further with a half-life of 100 µs, resulting in another oxoferrylintermediate F. On the other hand, if no electron is readily available at heme a, the cycle continues onanother path: Compound A decays to a species designated PM, also an oxoferryl adduct.

The main difference in the two routes is the electronic state of a tyrosine residue, Tyr244, in the vicinity.It has been determined that both P intermediates contain a O2- ligand, meaning that the O-O bond ofdioxygen has already been cleaved. This is only possible by a four-electron reduction. Three of therequired electrons can be accounted for by oxidation of the metal centers (Fe(II) to Fe(IV) and Cu(I) toCu(II)); the fourth electron, however, is from a different origin. Tyr244 has been identified as a redox-active cofactor, responsible for providing the fourth electron in the formation of intermediate PM, therebyassuming a transient tyrosyl radical character. If, however, the oxidation state of heme a is such that itcan donate an electron, the cycle goes on to PR.

The two oxoferryl intermediates both converge again at the same species F, albeit not with the samekinetic rate. To reach it, PM is reduced with an additional electron (more precisely it is the tyrosyl radicalthat is reduced), while this electron has already been absorbed by PR in the previous step.

Subsequent one-electron reductions occur via heme a as the electron-transferring cofactor. The so-calledfully oxidized species O is reduced in two steps via a semi-reduced intermediate E to the fully reduced a3complex, thus completing the cycle.20,21

17

Model Compounds forMononuclear Metalloenzymes

PART II

3Presently Available Models for Iron and

Copper Active Sites

The synthesis and characterization of biomimetic model compounds, aimed at replicating the featuresof an active site is a key tool in the elucidation of an enzyme and its mechanism. This chapter high-lights some of the more recent modeling approaches to non-heme iron and copper enzymes, laying thefoundations for the experimental work described in subsequent chapters.

3.1. Purpose and Significance of Model Compounds

Model compounds are comparatively small, low-molecular weight compounds that resemble or mimiccertain features of the active site of metalloenzymes. Ideally, model compounds reproduce the coordi-nation sphere of the metal center with regard to the ligands, structure, and oxidation state as closelyas possible. In some cases, model compounds are the first step on the way toward the development ofbiomimetic catalytic systems that catalyze reactions such as N2 fixation, CO activation and other feats ofenzymes.

As soon as the active site structure of a metalloenzyme has been elucidated, efforts begin to mimicthis structure in the form of small compounds. Biomimetic models can help in elucidating the enzyme’smechanism and provide a starting point from which to start the investigation of new catalysts. While ob-viously a model cannot reproduce more than the first coordination sphere, in most cases this is sufficientto exhibit a strong structural resemblance and often also enzyme-like activity.

Model Compounds lend themselves to a classification in two categories: Structural models aim to repli-cate primarily the geometric and electronic features of the active site. They provide constraints as towhat geometries are possible, what oxidation states are likely and how likely putative intermediates areformed. A lot of consideration has to be given to the steric architecture of the coordination sphere, oftennecessitating the design of special ligands.

Functional models, on the other hand, primarily try to imitate the (putative) enzyme mechanism. Ide-ally, a functional model can carry out a stoichiometric or even catalytic reaction that accomplishes thesame or a similar reaction as the original enzyme. Generally the particulars of the reaction, such as re-action rate and stereo control cannot be replicated in every detail, but the models still provide valuableinsight into and confirmation of a mechanism.22

Truly amazing examples of model compounds have been reported over the last decades. For instance,Collman et al. recently constructed a functional model of Cytochrome C oxidase, complete with theiron and copper center, capable of catalyzing the reduction of molecular dioxygen to water.19 A remark-able molybdenum-based center was reported by Schrock et al., capable of catalytically reducing N2 toammonia, albeit at low rates and with low turnover.23 A functional model capable of rudimentary pho-

19


tosynthesis was reported by Wieghardt already in the late nineties, mimicking a whole reaction cascade,rather than a single enzyme.24

Despite all these remarkable examples of the progress in biomimetic modeling, some features of en-zymatic reactions are still difficult to replicate. Especially the selectivity of enzymes is unsurpassed:Regarding substrate specificity, model compounds cannot compete with enzymes. In most cases eventhe best models are also unable to mimic the regio-, stereo-, and enantioselectivity. Additionally, truly‘life-like’ models of enzymes that reflect both structure and mechanism at the same time are rare.25

Much of the success of a particular metalloenzyme-inspired model is dependent on the ligands em-ployed. A lot of the work involved in the development of a particular model consists of designing andsynthesizing the right ligands that best resemble the structure and/or function of a particular center. Onlyafterwards can the ligand then be attached to the metal in the hope of arriving at a viable low-molecularweight model compound. In many cases the ligands have to be substituted with special sterically de-manding side-chains and complicated appendages that only in the final complex resemble the desiredactive site.

The following sections focus on recent model compounds for non-heme iron enzymes, especially cate-chol dioxygenases, and copper enzymes such as galactose oxidase.

3.2. Iron Enzymes: Modeling Catechol Dioxygenases

Catechol dioxygenases and their mechanism have already been introduced in section 1.2.2. A numberof structural as well as functional models have been constructed for both intra- and extradiol-cleavingdioxygenases.

A first functional model with both intradiol- and extradiol-cleaving activity was reported already inthe seventies by Funabiki et al., who used mixtures of pyridine and bipyridine as ligands to a Fe(III)center.26 The complex showed oxygenase activity upon reaction with 3,5-di-tert-butyl catechol, formingboth extradiol and intradiol cleavage products. While the exact structure of the compound is uncertain,this is nevertheless a first example of a functional model for catechol dioxygenases. However, this modelis primarily of historical interest since it incorporates hardly any features specific to the active site of theenzymes and has little or no tendency toward either of the cleavage reactions.

Subsequent modeling efforts have been more successful and a great number of elaborate model systemshave been developed.

3.2.1. Intradiol Catechol Dioxygenases

Structural Models

Since the iron center in the active site of native intradiol enzymes is coordinated by two His and two Tyrresidues, structural models tend to replicate this feature, aiming to reproduce the geometry as it is foundin the enzyme as closely as possible.

Starting already in the early 1980’s, model compounds primarily relied on the use of macrocyclic lig-ands of the salen-type (figure 3.1(a)) coordinated to ferric centers. The first crystal structures of suchstructural models were reported by Heistand et al.,27 in the form of a complex with a catechol substratealready coordinated to the center. In this case, however, the catecholate was found not to chelate themetal center, but instead as a monodentate κ-C complex.

Structure and reactivity investigations with the salen ligand and derivatives thereof have been carriedon to this day, for instance in the research of Mialane et al.28 in the late nineties, who used a salen-derived ligand with tertiary amine donors instead of imines. A rather recent and also the most ‘life-like’

20


structural model was reported by Fujii et al.29 in 2002. They employ a sterically extremely demandingmesityl-substituted salen-ligand (figure 3.1(b)) to force the metal center from a rather planar geometryinto a more trigonal bipyramidal one, as found e.g. in 3,4-protocatechuate dioxygenase (3,4-PCD).

OH

N N

HO

(a) Salen

OH

N N

HO

(b) Mes6-Salen

OH

N

HO

Me2N

R1R1

R2 R2

(c) Tripodal Diphenolate Ligand

Figure 3.1.: Tetradentate Ligands for Models of Intradiol Dioxygenases

Figure 3.2 shows a side-by-side comparison of the active site structures of 3,4-PCD and the Mes6-salenmodel complex. Interestingly, the chloro-analogon of this aqua complex shows the square pyramidalgeometry expected for the salen ligand. After treatment of this precursor with AgClO4·H2O , the chlorideligand is exchanged for H2O and a drastic structural change is observed. Depending on the externalproton concentration, the aqua ligand undergoes reversible deprotonation, thereby forming a hydroxospecies, which is also thought to be the ligand in the native enzyme under physiological conditions.Aside from the close structural similarity, this model also closely replicates the spectroscopic propertiesof 3,4-PCD.

Figure 3.2.: Comparison of the Active Site Geometry of 3,4-PCD and a Mes6-Salen Fe(III) Model Complex29

Functional Models

In spite of their close structural resemblance to the enzymes, the aforementioned model compounds showlittle to no catalytic activity. While some reaction with catechol substrates is frequently observed, reactionrates are very low and the products are quinones rather than cleaved hydrocarbons. Other compounds,while not as close to the enzymes structurally, show decidedly more catalytic activity.

The first functional models used a variety of tetradentate ligands, both macrocyclic and branched, with

21


different numbers of O- and N- donors, both heterocyclic and aliphatic. Examples include Nitrilotri-acetate (NTA), bis(2-pyridylmethyl)amino)acetate (BPG), 1,4,8,11-tetraazacyclotetradecane (cyclam) andmany more.

The first systematic study of ligand influence on the reactivity of the catalytic center was carried outby Que et al., using ligands such as those mentioned above. By varying the coordinating groups of theligands with regard to their electron-donating properties they could influence the Lewis acidity of theFe(III) center, thereby systematically modulating the catalyst’s reactivity. UV-Vis investigations of thevarious complexes and their catecholate adducts show that increasing the Lewis acidity of the centerdecreased (red-shifted) the transition energy of the LMCT absorption. This indicates that the t2g acceptororbitals of the metal center decrease in their relative energy level and approach the ligand donor orbitals.Consequently, the reaction rate and the product yield increase.

Increasing the Lewis acidity of the metal center by itself, however, is not the only explanation for in-creased reaction rate, or, indeed catalytic activity altogether. Replacing Fe(III) by Ga(III), for instance,results in complexes that have no activity at all. This leads to the conclusion that the intrinsic redox prop-erties of the metal ion are also crucial to the catalytic mechanism. A redox-active metal center can increasethe radical character of the bound catecholate by (partially) abstracting one of the electrons, resulting ina reduced metal and a semiquinonate radical species. The more pronounced radical character of the sub-strate facilitates subsequent reaction with triplet oxygen, which is otherwise inert toward electrons withpaired spins.8

Another approach to the functional modeling of intradiol-cleaving catechol dioxygenases was pub-lished in 2003 by Velusamy.30 They synthesized a series of Fe(III) complexes with tripodal chelate ligands(fig. 3.1(c)) employing an N2O2 donor set. Two of the complexes were resolved by X-ray diffractometry,one of them with a trigonal-bipyramidal iron center (R1=R2=CH3), the other one with a distorted octahe-dron (R1=H, R2=NO2). Contrary to the native enzyme, the former of the compounds showed no catalyticactivity, while the latter produced intradiol cleavage products. The difference in coordination geometry ofthe two complexes is not entirely evident, since the only difference in the ligands is a nitro-group insteadof a methyl residue on the two phenol moieties. Nevertheless unusual is the achievement of a trigonalbipyramid, the only other example of such a structure being the aforementioned complex by Fujii.

Predominantly, however, most functional models available so far, have only shown significant reactiv-ities when used in stoichiometric amounts. Models with true catalytic ability are much less common andhave not been studied in great detail. The few complexes examined under conditions of excess substratehave shown catalytic activity, but the reaction proceeds with significant yields only on a timescale of sev-eral hours. The faster-reacting compounds, on the other hand, show diminished selectivity and result in1:1 mixtures of intra- and extradiol cleaved product.8

3.2.2. Extradiol Catechol Dioxygenases

Modeling extradiol-cleaving enzymes has proven more difficult, and fewer models have so far been re-ported.

An iron(II)-tripyridyl amine catecholato complex has been reported as a structural model in good agree-ment with the crystal structure of the enzyme. This has given strong support to the assumption that thesubstrate binds as a monoanionic bidentate ligand. The other four coordination sites of the distortedoctahedral iron center are occupied by the nitrogen donor moieties of the ligand. Aside from this com-plex, however, very little has been reported on the synthesis of structural models for extradiol catecholdioxygenases.

22


Functional Models

While the native enzymes utilize an iron(II) center for the catalytic cleavage of catecholates, most func-tional models reported so far, are iron(III) complexes, and are fewer in number compared to intradiol-cleaving models. Complexes with ferrous iron react with oxygen to give the corresponding ferric species,without affecting the bound catecholate in any way. Since the metal center has a saturated coordinationsphere (bidentate catecholate and tetradentate ligand), molecular oxygen cannot coordinate to the metalcenter and single-electron oxidation is likely to occur via an outer sphere mechanism.

Some extradiol-cleaving activity was observed by Bugg et al. using a FeCl2 TACN (=1,4,7 triazacy-clononane) system in the presence of pyridine.31 While the selectivity of the catalyst was not exclusive,a 7:1 preference of the extradiol cleavage product was observed. The reaction proceeded with a varietyof substituted catechols, showing a higher activity for substrates with electron-donating groups and noobservable reaction for those with electron-withdrawing substituents. Since the presence of one equiv-alent of pyridine is required for the reaction to occur, the mechanism is likely to rely on a monoanioniccatecholate as mentioned in chapter 1. The fact that the monosodium salt of the catechol is also processedfurther supports this hypothesis. The use of macrocyclic ligands other than TACN or the substituion ofthe nitrogen donor moieties by oxygen, showed a significant drop in activity, while replacing Fe(II) byFe(III) resulted in a much lower tendency toward extradiol cleavage (2:1).

Some improvements have since been made to the TACN/Fe(III) system, for instance by Que et al. Byusing a Me3-TACN ligand in combination with the anionic catechol substrate and Ag+ salt, they wereable to achieve exclusively extradiol cleavage with near-quantitative yield.

OH

NMe2N

N

(a) Pyridyl Phenolate Lig-and (L)

NN

N

R

(b) Bis-pyridyl Ligand (L2)

Figure 3.3.: Monophenolate Model for extradiol cleavage32

More recently, Mayilmurugan et al. reported a novel tripodal iron(III) complex showing extradiol-cleavage capability.32 The group prepared a sterically hindered phenolate ligand (HL) with two aliphaticnitrogen donors and one pyridyl moiety. Fig. 3.3(a) shows the structure of HL. Coordination of L- to FeCl3yielded a complex of the composition [Fe(L)Cl2].

The angle of the Fe-O-C bond in the model (134.0◦) is very close to the angle in the equatorial tyrosineresidue in the enzyme (133◦). Also, the determined bond length of 1.889 angstroms for the Fe-O bondis similar to one Fe-Otyr distance in the enzyme (1.91 angstroms). Remarkably however, the two Fe-Otyr

distances in the enzyme itself differ by 0.1 angstroms (5.5%) from each other, which further emphasizesthe importance of the protein backbone in ligand alignment.

The group investigated the reactivity of the complex toward a diisobutylcatecholate (DBC2-) substratein various solvents in the presence of oxygen. The adduct [FeL(HDBC)Cl] was formed in situ by reactionof [FeLCl2] with H2DBC in the presence of two equivalents of triethylamine in methanol. Reaction withexcess oxygen for 48 hours yielded 17.8% of almost exclusively extradiol-cleaved products.

In quick succession to the publication of the aforementioned complexes, Visvaganesan et al. reporteda number of closely related model compounds.33 The group systematically investigated the properties ofbis-pyridyl substituted alkylamines 3.3(b) that were cis-facially coordinated to iron(III) for their structuresand catechol-cleaving activities.

23


OH

N N

HO

tBu

tBu

tBu

tBu

(a) Substituted Salen

N

NHHN

R

(b) TACN

OH

S N

HOH2N

(c) Tripodal Ligand

Figure 3.4.: Ligands for GAO Models

Many complexes with different alkyl and cyclo-alkyl residues were investigated and most of themshowed extradiol-cleaving capacities, whereby the selectivity of intra- vs. extradiol cleavage dependedon the nature of the substituent. All of the ligands coordinated cis-facially to the iron center, leaving twoadjacent labile coordination sites for substrate binding. Several X-ray structures of the complexes werealso determined and showed significant resemblance to the active site structure of 2,3-dihydroxybiphenyl1,2-dioxygenase.

With regard to reactivity and selectivity, their model compounds separate themselves into two groups.The ligands with R=-CH2CH(CH3)2, R=-CH(CH3)2, and R=cyclohexyl exhibit yields of≥60% of extradiolcleavage product, with a relative selectivity of 6:1 extradiol vs. intradiol. In contrast, those with R=-H,R=-CH3, and R=-tBu showed only yields of 30% and almost no selectivity (1:1).

The practical research on model complexes was based on some of the compounds mentioned aboveand will be discussed in greater detail in the following chapters.

3.3. Copper Enzymes: Models for Galactose Oxidase

A number of interesting biomimetic models for galactose oxidase have been reported in the last decades.Modeling the active site of GAO requires generation of a stable phenoxyl radical moiety and embeddingit in a coordination compound, which in itself is not trivial. The aromatic ring system needs to be stabi-lized by appropriate ortho- and para-substituents that either sterically prevent dissipation of the radical orconfer additional mesomeric stability to it.13

Model compounds for GAO successfully utilize some of the already mentioned polydentate ligands,such as salen, TACN, and tripodal ligands. Each class of model compounds will be briefly introduced.

Salen-based model compounds

The structure of the salen scaffold is shown in fig. 3.1(a). If the phenol moieties are substituted with bulkygroups in o- and/or p-positions, the formation of dimers can be prevented and phenoxyl radical speciescan be stabilized. Various salen ligands coordinate to Cu(II) centers and by varying the groups linking thetwo salicylidene moieties, the degree of flexibility and thus the distortion from a square-planar geometrycan be tuned.

The complexes can undergo two successive single-electron oxidations to a phenoxyl and subsequentlya bis-phenoxyl radical species. Obviously, in order to be in agreement with the native enzyme, the speciesmust be present in a Cu(II)-monophenoxyl state. For instance, a ligand reported by Thomas et al.34

is shown in fig. 3.4(a). The relatively stiff linking phenylidene linking moiety provides a square pla-nar coordination environment with a slight distortion toward tetrahedral geometries, thereby mimicking

24


the pyramid base in the active site of the native enzyme. The complex was characterized by opticalspectroscopy and cyclic voltammetry. Stepwise oxidation from [Cu(II)L] via [Cu(II)L·]+ to [Cu(II)L··]2+

showed essentially no change in the wavelength of the absorption maximum (445 nm), while the molarabsorptivity decreased with increasing degree of oxidation. In contrast, the magnetic properties of thecomplex undergo more significant changes. The reduced complexes show significant magnetic moments,as measured by EPR. One-electron oxidation to the mono-radical species results in spin-coupling of theunpaired electrons, making the compound EPR-silent. Subsequent oxidation to the diradicals in turnresults in a measured spin of S=3/2 due to ferromagnetic coupling of the three electrons.

Similar salen ligands have also been used in the synthesis of functional models. Wang et al.35 report atetrahedrally distorted Cu(II) complex capable of oxidizing benzyl alcohol to benzaldehyde with molec-ular dioxygen at room temperatures with turnovers >1000.

TACN-based model compounds

Another ligand scaffold suitable for the modeling of GAO is the 1,4,7-triazacyclononane macrocycle (fig-ure 3.4(b)) and derivatives thereof. The N donors of the cycle generally occupy one face of the metal’scoordination sphere, while possible substituents can coordinate to the vacant sites. GAO models gener-ally have one, two or three N-bound phenol residues that subsequently bind axially to the metal centerto give e.g. a distorted square pyramidal geometry.

One of the early models for GAO using TACN ligands was reported by Tolman et al.36 in the late 1990’s.The group prepared a series of phenolate-substituted TACN compounds, in some cases also incorporatingthe Car-S-C bond found in the enzyme. The complexes were investigated by optical spectroscopy, EPR,NMR and in some cases by crystallography. The resulting Cu(II) complexes were in some cases in goodagreement with the structure of the enzyme. Electrochemical characterization of the compounds showedthat they could all be oxidized to the respective phenoxyl radicals although the reaction was not in allcases reversible. This is attributed to spontaneous decomposition of the radical species by C-C bondcoupling processes involving the benzylic positions para to the phenoxyl oxygen. Those complexes thatcarried stabilizing tert-butyl groups, on the other hand, formed a reversible phenolate/phenoxyl redoxcouple. When comparing the complexes with the added alkylthio moiety to those without it, only smallshifts in the optical spectrum and redox potentials could be observed.

Tripodal Ligands

A variety of tripodal ligands have also been successfully used in the synthesis of biomimetic GAO models.Constrained only by the fact that the resulting coordination geometry should closely resemble a squarepyramid, any number of tripodal ligands are suitable, as long as they support a 5,6,6-membered chelatesequence.37 Therefore, an immense number of tripodal ligands, most of them with a central nitrogenatom have so far been reported, only a few examples of which will be mentioned here.

A ligand with close relevance to those studied in this thesis was reported by Ochs et al. in 200138

and is shown in fig. 3.4(c). Coordination of this ligand to Cu(II) resulted in the formation of a dimericcomplex, whose structure was resolved by X-ray crystallography. The two copper centers each residein a square pyramidal environment, bridged by two acetate-oxo bridges remaining from the Cu(OAc)2starting material. Additionally, each of the metals is coordinated by one tertiary and one primary amine,and the phenolate oxygen. For reasons not determined by the authors, the hydroxypropyl arm doesnot coordinate to the metal center and remains protonated. While from a structural point of view, thiscomplex is not a suitable model for galactose oxidase, it still provides the necessary framework for thedesign of related compounds more similar to GAO in their structures.

25

4Synthesis of Model Complexes

The culmination of the theoretical elaborations in the preceding chapters is to now put them in a prac-tical context and apply them to an actual research problem. During a three-week practical project withthe Mösch-Zanetti group at the University of Graz, the synthesis and investigation of various enzyme-inspired iron(III) and copper(II) complexes led to promising results.

In the course of 15 research days a total of 22 substances, ligands as well as metal complexes, weresynthesized, characterized and have provided valuable insight regarding the preparation and propertiesof compounds previously unreported. Inspired by the work of Mayilmurugan32 and Gibson39 a seriesof aminophenolate complexes of Fe(III) and Cu(II) were synthesized with the aim of finding model com-pounds for iron- and copper-dependent metalloenzymes.

The remainder of this thesis is an account of the research carried out between September 1st and 19th,2008 under the supervision of Prof. Dr. Nadia C. Mösch-Zanetti and Mag. Martina Judmaier at the In-stitute of Chemistry: Inorganic Chemistry, University of Graz. This chapter outlines the research goalspursued and the methods attempted to reach them. The necessary background information as well asa cursory overview of the employed procedures and methods are also given. The following chapter de-tails and discusses the results obtained and summarizes what was learned and what still remains to beinvestigated. The appendix contains a detailed account of all experiments carried out and the resultingdata.

4.1. Research Goals

The research project set out with a number of objectives, aiming to synthesize and investigate severaldifferent monophenolate-coordinated Fe(III) and Cu(II) complexes, based on active-site geometries of therespective metalloenzymes.

Preparation of New Thioether-substituted Aminophenolate Donor Ligands

OH

SN

CH3

D

n

2-n

Figure 4.1.: Ligand Structure

As an addition to the set of already available model ligands, as describedin chapter 3, a number of additional ligands shall be synthesized. Fig. 4.1shows the general structure of the ligands, where D is a donor moiety, either-OMe or -NEt2, and n is 1 or 2. Preparation of the ligands should proceed viastandard methods, starting from 4-methylphenol by methylthiomethylationand subsequent Mannich amination.36 Characterization of the products anddetermination of purity shall occur by 1H-NMR spectroscopy. The availableligands will then be brought to reaction with metal salt precursors, aiming toisolate viable complexes.

26


Determine the Feasibility of Metal Complex Synthesis

M

O

N

S

D

D

ClCl

Figure 4.2.: Structure of Complex

In analogy to the macrocyclic NNN Cu(II) and tripodal complexes previouslyreported (fig. 3.4(b) and 3.4(c)), synthesis of Fe(III) and Cu(II) coordinationcompounds with tri- and tetradentate branched ligands shall be attempted.Presumably, the resulting complexes have octahedral geometries (fig. 4.1),the remaining coordination sites occupied by the chlorides remaining fromthe metal salt.

Evaluate and Refine the Employed Methods

Various synthetic routes lend themselves for the coordination of penolate lig-ands to the metal salts. Depending on whether the ligands are deprotonatedby an additional base such as triethylamine or instead their sodium or potas-sium salts prepared, the yield is likely to vary. Therefore, synthesis will be approached via multiple routestaking care to evaluate and discontinue unsuccessful strategies and improve successful ones. Also, theproducts will be investigated for air and moisture sensibility and procedures adapted accordingly.

Characterize Resulting Products as Exhaustively as Possible

The obtained products will examined closely by all suitable methods. Since the metal centers are param-agnetic, ordinary nuclear resonance spectroscopy is unlikely to provide results. However, methods suchas mass spectrometry, IR spectroscopy, and special NMR techniques such as Evans’ Method can providea lot of information about the prepared compounds. Especially the latter is suited for the investigation ofparamagnetic compounds, since it allows the determination of the number of unpaired electrons. Crys-tallization of the products and X-ray diffraction measurements shall also be attempted.

4.2. Materials and Methods

4.2.1. Preparation of the Ligands

OH OH

S

1 RK01

Me2S/Cl21h RT/18h reflux HCHO, MeOH (reflux)

OH

SN

CH3

D

2-n

n

HN

H3C D2-n n

RK02-5

Figure 4.3.: Ligand Preparation (D=–OMe or –NEt2; n=1 or 2)

Starting from 4-methylphenol (p-Cresol, 1), precursor RK01 was obtained by methylthiomethylation(procedure see pg. 34 and ref. [36]) using chlorine gas and dimethyl sulfide. After extraction and sol-vent removal, the crude product was obtained as a brown oil, which was purified by destillation. Theprecursor was obtained with 45% yield in high purity as determined by 1H-NMR.

The different amine donor moieties were attached by Mannich reaction using aqueous formaldehydein methanol (see pg. 35). The ligands were obtained as viscous oils ranging in color from yellow to brownwith yields between 60 and 90%. Characterization by 1H-NMR confirmed the formation of the desiredproducts.

27


4.2.2. Preparation of the Complexes

Two methods lend themselves to achieve the actual coordination of the phenolate ligand to the metal salts.Both the deprotonation by an external base and the preparation of the potassium salts have differentadvantages and disadvantages. While the former does not require strictly inert conditions and doesnot make use of hazardous materials, triethyl ammoniumchloride forms as a byproduct and has to beremoved. The latter method, on the other hand, relies on potassium hydride and therefore requires alloperations to be carried out in a dry and inert atmosphere. Additionally, special care must be taken whenhandling and storing the hydride.

Anhydrous FeCl3 and CuCl2 were chosen as metal salt precursors. The former is relatively stable asblack crystals under ambient conditions and is readily soluble in methanol and tetrahydrofuran. Thelatter is a brown crystalline solid that needs to be stored in a dry environment and is also soluble in THF.

For reasons of clarity, the ligands and complexes will be referred to according the nature of the donormoieties. E.g. -NO refers to the aminophenol ligand with one –OMe substituent, while -NNN refers to thespecies with two –NEt2 residues.

Method I: Deprotonation with Triethylamine

The procedure was first attempted with a number of differently substituted aminophenol ligands, thosepreviously synthesized as well as some that had already been prepared. The most basic approach con-sisted of simply adding a stoichiometric mixture of ligand and triethylamine in methanol to a solutionof FeCl3 and refluxing for 18 hours. Upon addition, an instant color change was observed, differentdepending on the ligand used.

In some cases a solid precipitated from the solution, either already at room temperature or after refrig-eration. Characterization of the obtained solids by MS, however, showed no encouraging results and thesubstances all turned brown quickly, presumably due to oxygen exposure.

For this reason, the preparation was attempted again for one ligand (-NNN) in inert atmosphere (seepg. 39). In this case, a dark-blue solution was obtained and after refrigeration a blue crystalline solidprecipitated and could be filtered off.

The procedure was also applied to the preparation of the Cu(II) -NO complex. The product was ob-tained as a green microcrystalline solid.

Method II: Preparation using Potassium Salts

Since the preparation using triethylamine was not very successful in yielding viable products, anotherapproach was attempted instead. The procedure consisted of preparing the potassium salt of the ligand,resulting in a potassium phenolate (see pg. 37). The ligand was dissolved in THF and a slight excess ofpotassium hydride added. The solution was stirred for 18 hours while the evolution of H2 gas persisted.The mixture was filtered over celite and and the solvent removed in vacuo. The potassium salts wereobtained in yields of roughly 60%.

A disadvantage of this method is the fact, that all steps need to be carried out under extremely dryand oxygen-free conditions. Any residual contamination by water destroys the hydride, resulting indiminished yields and possible hazards from evolving hydrogen gas.

The ligand salts were subsequently redissolved in THF and a stoichiometric amount of FeCl3 wasadded (see pg. 38). Again, a color change was observed each time, different for each of the ligands.After stirring for 18 hours, the solid KCl precipitate was filtered over celite and the solution dried invacuo. Most of the complexes did not crystallize and remained as amorphous, slightly oily residues.

28


Although this procedure was not entirely successful, either, it does have some advantages. Since theKCl by-product is only very slightly soluble in THF it can be easily removed by filtration. Compared toother methods that use triethylamine, the salt can therefore be quantitatively be removed and does notinterfere with subsequent purification and crystallization efforts. Also, since all the steps of the proce-dure are carried out under inert atmosphere, oxygen and water cannot act as potential contaminants anddestroy the formed compounds. With additional time to refine and re-attempt the procedure, successfulpreparation of the complexes is very likely.

4.3. Characterization

4.3.1. Ligands

Characterization of the identity and purity of the ligands was carried out by established methods, pri-marily 1H-NMR spectroscopy. The samples were dissolved in CDCl3 and measured at 360 MHz with aBruker AMX 360 spectrometer. NMR Data of the four ligands RK02 – RK04 as well as the precursor RK01are reported on pages 34 and 35. All peaks were unambiguously assigned to the structural moieties, theirchemical shifts being in the expected range and constant for corresponding protons in different com-pounds. Volatile impurities were removed in vacuo, resulting in virtually no additional impurity peaks,and if so only with negligible intensities.

Since the determined purity was sufficient for the subsequent synthetic steps, no further purificationwas carried out. No major side reactions are expected for the Mannich amination and the unreactedprecursor is volatile enough to be removed under reduced pressure.

Further determination and quantification of impurities could be carried out e.g. by liquid chromatogra-phy or a number of other separation techniques. Purification is probably possible by column chromatog-raphy or liquid-liquid extraction, but was not attempted.

4.3.2. Coordination Compounds

Since all the complexes are expected to be paramagnetic, methods such as NMR spectroscopy are not suit-able for characterization. A number of other techniques were attempted instead, with a varying degreeof success.

All complexes and also the potassium salts of the ligands were characterized by mass spectrometry.None of the Fe-complexes prepared under non-inert atmosphere showed any masses or fragments in-dicative of a complex. In fact, some of them showed no significant fragmentation at all and were notsufficiently ionized. This may be an indication that the complexes are unstable in air and decayed to fer-ric oxide or a related species, that could subsequently not be measured by MS. Spectra of the potassiumsalts were also inconclusive.

A viable MS spectrum was obtained from compound RK19, as shown in fig. 4.5. Fragmentation sug-gests the formation of the complex, which is also supported by subsequent IR mearsurements. All MSmeasurements were carried out using an Agilent 597-3 MSD direct probe mass spectrometer using EIionisation.

The [Fe(-NNN)Cl2] complex (RK20) was additionally characterized by IR spectroscopy on a Perkin-Elmer FT-IR 1725X spectrometer. Additionally, spectra of pure FeCl3 and the potassium salt of the ligand(RK16) were also recorded. Fig. 4.4 shows all three IR spectra next to each other. Despite some baselinedrift in the upper two graphs, the spectra still give some information about the isolated product.

29


4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0

Trans

miss

ion

W a v e n u m b e r ( c m - 1 )

Figure 4.4.: IR Spectra of RK20 (solid line), RK16 (dashed), and FeCl3 (dotted)

Figure 4.5.: Mass spectrum of compound RK19

30

5Results and Discussion

5.1. Tri- and Tetradentate Ligands for Versatile CoordinationCompounds

Fig. 5.1 shows the four synthesized ligands. While examples of similar compounds abound in recentpublications, these ligands as such are previously unreported. They have been unambiguously isolatedand characterized. They are easily accessible by two-step syntheses, using only common and widelyavailable starting materials.

The ligand design is a blend of tetradentate ligands already published by Gibson39 and Tolman36 andincorporates a number of structural features that makes them suitable as model compounds. The -NOOand -NNN ligands expand on a model for galactose oxidase that uses a macrocyclic salen-based donorarrangement. The branched acyclic structure makes these ligands more flexible and enables a greaternumber of possible coordination geometries.

Varying the number and nature of the donor moieties allows the examination of structure-stabilityrelationships in combination with different transition metals. Among any number of conceivable appli-cations, they expand the number of available framework ligands for both galactose oxidase and catecholdioxygenases. While the previously published models for extradiol catechol dioxygenases employ at leastone aromatic (pyridyl) N-donor element, the structure and reactivity of purely aliphatic phenoxyamineFe(III) complexes has hardly been addressed so far.

Preparation of these four compounds is therefore the initial step toward the preparation of novel modelcompounds for enzymes and numerous other coordination compounds. The four new ligands reportedhere constitute an addition to the set of available ligands for bioinorganic and complex chemistry ingeneral and expand the range of starting materials for the synthesis of metal complexes.

OHS

RK02

NO

(a) -NO

OHS

RK03

NN

(b) -NN

OHS

RK04

NN

N

(c) -NNN

OHS

RK05

NO

O

(d) -NOO

Figure 5.1.: Novel Tetradentate Ligands

31


Fe

O

N

S

O

O

ClCl

(a) Facial Geometry

O

N

S O

OFe

ClCl

(b) Meridional Geometry

Figure 5.2.: Possible Geometries for [Fe(-NOO)Cl2]

5.2. Spectroscopic Evidence for a New Monophenolate Iron(III)Complex?

In addition to the successful preparation of the ligands, compelling evidence for a new monophenolate-NNN Fe(III) complex can be reported. Synthesis of the complex was achieved by both methods men-tioned above, whereby the triethylamine-based approach under inert atmosphere (RK20) produced acrystalline solid when the potassium-salt precursor only led to an amorphous product (RK19).

RK20 was characterized by infrared spectroscopy (figure 4.4) and shows evidence of ligand coordina-tion. Ligand absorption bands match those found in the isolated complex. This is substantial evidence,since the uncoordinated FeCl3 shows no absorption at all below 1600 cm-1 and therefore does not interferewithin the fingerprint range. Any uncoordinated ligand, on the other hand, would have been removedby washing. It can thus be inferred with some likelihood, that the desired complex has indeed formed.

Supporting evidence for this is provided by the mass spectra of the complexes RK20 and RK19. Whilethe former is not of optimal quality, numerous peaks indicative of successful product formation arepresent in both. A fragment corresponding to the unbound ligand is rather prominent in the RK19 spec-trum, together with a [Fe(-NOO)] fragment and a [Fe(-NOO)37Cl2] fragment, corresponding to the finalcomplex. This lends additional support to the fact that the complex has been isolated as assumed.

The aforementioned methods unfortunately provide no information about the nature of the coordina-tion, the structure and possible isomers of the complex. Figure 5.2 shows two likely coordination isomersfor the -NOO complex. 5.2(a) assumes a cis-facial coordination of the NOO donor moiety, while 5.2(b) isa meridional arrangement. The latter structure is in agreement with a complex reported by Gibson39 fora very similar Cr3+ complex, as determined by X-ray diffraction. The corresponding Fe2+ complex of thesame ligand, was found to be a bis-µ-Cl dimer. A related complex by Tolman36 employed a macrocyclicligand, thereby enforcing a facial coordination analog to the former proposed structure.

There is, however, no practical method short of X-ray crystallography that could elucidate the precisecoordination geometry. Attempts to obtain X-ray grade crystals of the compounds, have so far beenunsuccessful, and hence the nature of ligand coordination remains undetermined. Promising attempts forcrystallisation consisted of slow evaporation of various solvents as well as diffusion of unpolar solventsinto a solution of the complex. Diffraction measurements on the prepared compounds are definitely notout of reach, and would ascertain the identity and structure of the complexes.

5.3. Outlook

While a research period of three weeks is rather short to yield significant breakthroughs, promising firststeps toward the preparation of novel coordination compounds have been made. Appropriate follow-ups would consist of further characterization of the obtained compounds, refinement of the procedures,

32


structure determination, and eventually reactivity studies.An aspect of the complexes that has yet to be investigated are their magnetic properties. A first approx-

imation of the number of unpaired electrons/the magnetic moment could be measured with ordinaryNMR using Evans’ method. Subsequent SQUID measurements could further elucidate the spin statesof the metal center’s electrons. The electronic properties of the compounds also demand further atten-tion. Cyclic Voltammetry (CV) measurements would provide insight into redox potentials and reachableoxidation states of the complexes, thereby setting the first step toward studying reactivity and catalyticproperties. Crystallographic str

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