LIST OF ABBREVIATIONS

Materials

Boctert-butyloxycarbonyl Boc-His(Boc)-OH . DCHA -

acetonitrile MeOH -methanol MES -2-(N-morpholino)ethanesulfonic acid NMP -N-methyl-2-pirrolidone NBTnitrotetrazolium Blue chloride NPP -4-nitrophenyl phosphate NP -4-nitrophenol nta -nitrilotriacetic acid SOD -superoxide dismutase TEA -triethylamine

INTRODUCTION

COORDINATION CHEMISTRY OF BASIC TRIPODAL LIGANDS

Tripodal ligands are specially structured molecules with three coordinating arms, containing several (usually at least three) donor groups. Due to their structural properties, they are extensively used in coordination chemistry as complexing agents for various transition metal ions. The key to their versatile applicability is the tripodal scaffold, which is able to organize the donor groups in space, leading to strong and controllable metal binding. Metal complexes of tripodal ligands usually possess elevated thermodynamic stability due to the forming fused chelate rings, which, depending on the denticity and symmetry of the ligand, may also help stabilize several coordination geometries. Targeted functionalization of the tripodal ligand allows the fine-tuning of metal binding, and also the development of additional functions, e.g. molecular recognition sites, secondary (allosteric) metal binding sites or substrate binding sites, etc.

For these reasons, metal complexes of tripodal ligands are targets of scientific interest in many fields, such as metal sensing [1], metalloenzyme modeling [2][3][4], stabilization of reactive species and intermediates [5,6] or development of molecular motors [7]. Tripodal ligands have recently risen the attention of supramolecular chemistry, since they present exceptional tendency to self-assembly creating metallocages with colorful compositions [8], hydrogels [9] or other mesophase structures [10].

With respect to the structure of tripodal ligand complexes, the center of the tripod is of great importance: it can be either a coordinating, trivalent heteroatom itself (such as nitrogen, phosphorous or boron) or a functionalized central group (e.g. benzene or cyclohexane platforms). Rigid scaffolds as benzene tend to stabilize unusual geometries, while a tertiary amino center provides flexibility to the ligand, often resulting oligomerization. These features, of course, are also dependent on the quality of the donor groups and the metal ion, and the length of the tripodal arms. presents examples for some basic, extensively described and used tripodal ligands. The structure of the mentioned basic tripods, having one donor group per arm in a chelating position, normally allows tri-or tetradentate binding to a transition metal ion, and the free coordination sites can be occupied by solvent molecules or counter ions. With respect to geometry, the quality of the metal ion, the solvent and the flexibility of the ligand together determines coordination number and thus the structure of the complexes: while rigid molecules like tach usually provide a relatively fixed, trigonal-based structure for metal ions, more flexible ligands like tren or nta may stabilize more than one type of geometry.

Tripodal ligands can be classified by many features: their symmetry (identical or different arms), the chemical property of the pendant donor groups (aromatic or aliphatic), the length of the arms, the type of the donor atoms, denticity, etc. Since a main aspect of this work is to compare the C-and N-terminally functionalized histidine-containing tripodal ligands, the N-and O-donor tripodal ligands are briefly reviewed below, focusing on the two most important target molecules, tren and nta, which were used as base tripodal scaffolds for the studied ligands.

N-donor tripodal ligands

Tripodal ligands containing N-donor atoms have enormous literature background, which denotes their high significance in coordination chemistry [11][12][13][14]. Being the most frequent donor group of biorelevant ligands, tripods with nitrogen donor functions are extensively researched in bioinorganic chemistry, thus their coordination properties towards copper(II), zinc(II) and iron(II/III) ions are studied in details.

The most widely investigated N-donor tripodal ligand is tren, tris(2-aminoethyl amine). It was first synthesized by Eugen Ristenpart in 1896, as trihydrochloride salt, in the reaction of 2-bromoethylphtalimide and ammonia, followed by deprotection with HCl [15]. Tren has four amino groups, of which the three primary amino nitrogen can be protonated between pH 2-11, therefore only those can be determined potentiometrically (pK NH 3 + = 8.43, 9.47 and 10.17 at 25 • C, in 0.1 M KCl) [11]. The low basicity of the tertiary amino nitrogen (pK < 2) is due to the relatively close proximity of the primal amino groups.

The aptitude of tren for chelating metal ions has been discovered soon after its first successful synthesis; metal complex formation in presence of tren has been studied within various conditions ever since. Most of its 3d metal complexes have been characterized in solution and solid phase as well. Metal coordination by tren results in thermodynamically favored, 5-membered, fused chelate ring system, which prevents classical fourcoordination (tetrahedral, square planar) geometries, and causes the formation of distorted five-coordinate complexes instead (Figure 1.2). The scale of this distortion is described by the  value (0 <  <1), where the two extrema correspond to the symmetric trigonal bipyramidal ( = 1) and the symmetric square pyramidal ( = 0) geometries [16]. In most of the cases, the central donor atom occupies the apical position, and the fifth coordination site is taken by a rapidly exchanging ligand such as water molecule or background/counter ion. In presence of certain metals (e.g. Co(III) ions) and/or co-ligands, tren may form octahedral complexes in a [M(tren)(X)(Y)] n+ manner, in which case two stereoisomers can be distinguished, p-and t-isomers, often having very different physicochemical properties. In case of asymmetric tripods (bearing arms with different length), this isomerism can become more complex, depending on the position of the longer/shorter arm with respect to the X and Y ligands. 4d and 5d metal complexes of tren are of lesser significance as compared to 3d metal complexes, reflecting their strong preference for square planar geometries, which is disfavored by tren. Therefore, hypodentate coordination is not rare in such 4d/5d metal-tren complexes [11].

Tren forms the most stable ML-type metal complexes among tripodal tetramine ligands, due to the optimal size of chelate rings (5-membered) around the metal ion. Tripods with longer arms result in less favorable chelate systems, therefore substitution of aminoethylamine arm(s) with aminopropylamine moieties causes a gradual decrease in stability. Copper(II) complexes of the 3-aminopropyl-bis(2-aminoethyl)amine (baep), 2-aminoethyl-bis(3-aminopropyl)amine (abap) and tris (3-aminopropyl)amine (trpn) ligand series convincingly represent this tendency: logK Cu(tren) (= 18.5) > logK Cu(baep) (= 16.55 [17]) > logK Cu(abap) (= 15.98 [18]) > logK Cu(trpn) (= 13.117 [19]).

O-donor tripodal ligands

Tripods bearing oxygen donor groups are not as profoundly studied as the N-donor counterparts. Oxygen containing functional groups are mostly present as carboxylic or hydroxyl groups, beside additional nitrogen functions in many cases.

The most important representative of O-donor tripodal ligands is the nitrilotriacetic acid, generally abbreviated as nta. Nta was synthesized from ammonia and chloroacetic acid in 1862 by Heintz [20], but its strong metal sequestering properties were not discovered until the 20 th century. Nta possesses four (de)protonable functional groups, yet only three can be determined in aqueous solutions: pK 1 = 1.65, pK 2 = 2.94 and pK 3 = 10.33 at 20C [21], of which the first two corresponds to the carboxylic moieties, while the last pK is the result of the increased basicity of the tertiary nitrogen, most probably due to intramolecular hydrogen bonding between the N tert -H and a deprotonated carboxylic O.

Similarly to tren, nta binds most metal ions in a tetradentate way, resulting highly stable mononuclear complexes (see Table 1.1). In case of most metal ions, the great stability of the complexes does not allow direct potentiometric determination, another competitor ligand (e.g. tren) must be used to reach full decomplexation of nta. Table 1.1. exhibits a comparison in stability constants of ML complexes of tren, nta and EDTA. [11], nta [22] and EDTA [23] The geometry preference of nta is interestingly much less straightforward than that of tren. For example, as described earlier, tren forms 5-coordinate [M(tren)(H 2 O)] n+ complexes with 3d transition metal ions, while nta-metal complexes are usually thought of as octahedral species, in a [M(nta)(H 2 O) 2 ] n+ manner. In the last fifteen years, however, experimental and theoretical studies revealed that the second water molecule is coordinatively weakly bound. This also goes for other [M(nta)L 2 ] n+ complexes; they easily lose a monodentate L ligand to result the five-coordinate, trigonal bipyramidal complex [24]. However, this process is highly dependent on the nature of the co-ligand; bidentate ligands or steric hindrances (e.g. immobilization in IMAC chromatography) suppress its significance.

TRIPODAL LIGANDS FUNCTIONALIZED BY PEPTIDE COUPLING

Although functionalized tripodal ligands and their metal complexes are extensively studied for various goals, only a relatively small group of these products contains amide or peptide functions. This might be partly due to the inevitable technical difficulties regarding the synthesis of these compounds: the peptide coupling has to take place in solution phase (except for a few special solid-phase techniques [25,26]), in presence of at least one coagent (coupling agent), therefore further purification step(s) are required. Peptide-type ligands are often not easy to handle in solid state due to e.g. hygroscopic properties even as salts; furthermore, tripodal peptides, especially on benzene or cyclohexane-type platforms and/or bearing aliphatic side chains have been shown to act as hydrogels [9]. The few examples of peptide-type tripods, however, generally present some interesting properties of biological/biomimetic relevance, proving the worth of this direction in bioinorganic researchdespite all preparation-related hardship.

The advantages of peptide-type tripodal ligands in coordination chemistry have been recognized in the last decades: they proved to be useful in e.g. the development of allosteric binding cavities in supramolecular assemblies [27], targeted metal chelation [28], structural or functional enzyme modeling [25,29], and catalytic applications [30]. Herr and co-workers reported equilibrium and structural study on zinc(II) complexes of two histidine containing tripodal peptides (N(BzGly*ValHisOMe) 3 and N(BzGly*ValHisSerOMe) 3 ); both were found to be structural mimics of the active center of carbonic anhydrase enzyme [31]. Ichikawa's research group synthesized a tren-based tripodal ligand containing benzyl-protected histidyl moieties, and described the complex formation properties in presence of Zn(II) ions. Beside the imidazole-coordinated complexes, they found evidence of 'extra' deprotonated species above pH 7, which was attributed to mixed hydroxo complexes, and was found to possess considerable hydrolytic activity towards activated phosphoester substrates such as BNPP and TNPP (bis and tris(4-nitrophenyl) phosphate) [32]. Later on, the C-terminal analog of this ligand has been prepared as well, but despite the presence of a OH --containing 'extra' deprotonated species above pH 6, hydrolytic activity was not detected in this ligand system [30].

In the last decade Delangle's research group studied several cysteine containing ligands on, among others, tripodal platforms such as nitrilotriacetic acid (nta), in order to create efficient chelators for Cu(I) and other soft-type (potentially toxic) metal ions [28,[33][34][35][36]. Vahrenkamp et al. also studied histidine and cysteine containing tripods with benzene-1,3,5-carboxylic acid and tris(carboxyethyl)-nitromethane platforms; the zinc(II) complexes of these compounds showed high tendency towards oligo-and polymerization [29].

Some copper(II) complexes can also be found in the literature of peptide-type tripodal ligands: Albada and co-workers were able to build a structural model of type-3 copper(II) binding sites using a triazacyclophane (TAC) scaffold functionalized by histidine amino acids: oxygen uptake and reactivity of the oxygenated intermediate have also been shown in H 2 DTBC oxidation [25]. Branched peptides investigated by Szyrwiel and colleagues can be considered as close relatives of tripodal peptides; theyespecially the trihistidyl compound 3Hwere shown to possess remarkable binding affinity towards Cu(II) ions [37,38]. They also described a Asp-functionalized ligand, which forms catalytically active copper(II) complexes for electrocatalytic water oxidation [39]. As example for non-histidyl compounds, a tren-based alanine ligand has been synthesized by Mohamadou et al., and characterized in presence of Cu(II) and Ni(II) ions. Interestingly, formation of dinuclear copper(II) complexes has been detected at metal excess, which, beside UV-Vis and EPR analyses, was also confirmed by ESI-MS results [40].

HISTIDINE CONTAINING PEPTIDE LIGANDS TOWARDS COPPER(II) AND ZINC(II) IONS

Histidine plays an essential role in the mechanism of the majority of native enzymes and metalloproteins, participating in e.g. proton transfer, metal or substrate binding. The key to its utmost importance in biological systems is the imidazole side chain, which possesses a highly basic (pyrrolic) N1 nitrogen, and an easily protonable N3 (pyridinic) nitrogen. The deprotonation constant of the conjugate acidic form of histidine is pK ~ 6, which provides an ideal acid-base catalytic site in peptides and proteins, meanwhile transition metal ions can easily exchange the N3-H proton, providing a strong metal-ligand interaction in the physiological pH range. Metal binding of His residues is further improved when occupying a terminal position, although this binding mode has lower importance in native proteins. Occasionally, the imidazole group can act as a bidentate ligand, serving as a bridge between two metal ions, as e.g. in the active center of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) enzymes. In summary, the presence of histidine(s) in a peptide sequence not only alters but usually determines the metal binding properties.

The role of histidines in the peptide chain

The imidazole group of histidine amino acid inside the peptide chain enables metal binding at relatively low pH, depending on the nature of the metal ion. Coordination properties of imidazole-coordinated complexes are highly dependent on many factors [41]: for example, the number and position of the histidyl moieties in the peptide, the presence of other potentially coordinative side chains such as carboxylic groups (Asp, Glu), thiol groups (Cys), or the presence of free termini. If the peptide contains separated His amino acids in the sequence (proximity in the chain > 3 amino acids), the simultaneously coordinating N im groups allow the formation of macrochelate structures in the acidicneutral pH range, as it is frequently observed for large natural peptide fragments as well, including prions or amyloid- peptides [42]. work in our laboratory [43]).

With increasing pH, the multiimidazole environment competes less and less with (1) the metal-induced deprotonation of the backbone amide nitrogens, or (2) the (partial) hydrolysis of the metal ion (Figure 1.3). The former process occurs mostly in metal complexes prone to form square planar geometries, such as Ni(II), Cu(II) and several 4d and 5d metals. Histidine can facilitate the amide coordination to the metal center by acting as anchoring group: a coordinating imidazole N3 atom forms highly stable six-membered chelate ring with a neighboring amide N -towards the N-terminus. The coordination of the next amide nitrogen occurs co-operatively in almost all cases, creating a new fivemembered chelate ring. In this fashion, two or three amide nitrogens can be present in the coordination sphere of the metal ion beside the anchoring group. Without another characteristic driving force of coordination, this process takes place already below neutral pH for most copper(II)-oligopeptide complexes, around pH 8 for Ni(II) complexes and at extremely acidic pH for palladium(II) complexes [42]. Another result of N -coordination in peptides containing separated histidines is the formation of multinuclear species, since each His provides an individual {N im ,xN -} type binding site.

Hydrolysis of the metal ion, i.e. the deprotonation of coordinated water molecule(s), occurs in peptide complexes where amide nitrogen coordination is not an option. Unsaturated coordination sphere generally facilitates the process, but in many cases, such as in Zn(II) or Fe(III) complexes, hydroxide molecules are able to force out donor groups from the coordination sphere, or trigger oligomerization, resulting insoluble mixed hydroxo oligomer formation. In absence of additional stabilization, precipitation is usually observed at mildly basic pH [41]. The stability of His-containing peptide complexes are generally increased if His occupies special position(s) in the peptide chain: terminal His, NH 2 -X-His, NH 2 -X-X-His and alternating H(XH) n type sequences provide elevated stability for their metal complexes compared to terminally protected, separate His-containing peptide species [41]. Figure 1.4. depicts the dominant binding modes in these peptide sequences for Cu 2+ ions. Peptides containing such structural motifs often serve as target ligands in biomimetic studies.

The role of terminal histidines

Terminally unprotected peptides generally form complexes with relatively high thermodynamic stability, since -NH 2 and -COO -functions can behave as anchoring group in metal binding. Histidine in the terminal positions, especially located at the N-terminus, further enhances the observed complex stabilities. While C-terminal histidine mainly facilitates amide deprotonation, a N-terminal imidazole forms highly stable six-membered chelate ring with the terminal -NH 2 group, creating a histamine-type binding mode (Figure 1.4, structure (1)), which dominates in a very wide pH range. The monocopper(II) complex of the simplest N-terminal His-peptide, His-Gly, is dominant between pH 3.7 and 6, while at ligand excess the bis-histamine-type CuL 2 complex (Figure 1.5, left) does not deprotonate until pH ~ 10.5 [44]. Both analogous Zn(II) complexes form predominantly until basic pH, however their log values (log ZnL = 5.07, log ZnL 2 = 9.54 [45]) are significantly lower than that of the copper(II) complexes (8.85 and 15.06, respectively). It is worth mentioning that, even though Zn(II) supports the symmetrical octahedral geometry, tris-histamine-type complexes in Zn(II)-His-Gly system are not produced at any metal-to-ligand ratio. The smallest model of the C-terminal His containing peptides, namely the Gly-His ligand, also presents a characteristic metal binding ability (Figure 1.5. right). Due to the formation of a (5,6)-membered fused chelate ring system, the deprotonation constant of the C-terminal amide group decreases significantly: CuL  CuH -1 L + H + pK = 3.39 [44] ZnL  ZnH -1 L + H + pK = 6.73 [45].

In fact, this is one of the very few cases when Zn 2+ ions are capable of promoting the deprotonation of amide nitrogens in peptides; aside of Gly-His, zinc(II) complexes of Ala-His and Ala-His also present similar coordination behavior [46].

ENZYME MIMICKING USING METAL COMPLEXES

A very relevant direction of modern bioinorganic research is the design of lowmolecular models of metalloenzymes and -proteins. Native enzymes are the most efficient catalysts, accelerating biochemical reactions by several thousand times. This catalytic performance, along with their remarkable selectivity, has become highly desirable with the rapid development of biochemical industry. The usage of native enzymes in biochemical production, however, poses several difficulties: they are expensive, their structure is highly sensitive to the reaction conditions (pH, T, concentration of substrate(s), co-agents, product(s), etc.) causing eventual degradation, and their recovery is also problematic. Therefore, there is a constant need for the development of enzyme alternatives possessing similar (or at least sufficient) catalytic properties, more robustness, less expensive procurement; but they should also be environmentally friendly, and preferably recyclable.

The key to the tremendous efficiency of enzymes is their well-defined three dimensional structure, perfected by millions of years of evolution, in order to function in a very specific environment in living organisms. If we drastically change this environment, the tertiary structure might get damaged, leading to the loss of catalytic activity. The utmost challenge of enzyme mimicking is to substitute the great and sensitive polypeptide structure with a simpler, robust organic ligand, which can provide similar coordination environment to the active center of the metalloenzyme. It is clear that there is no general solution for this problem; each different enzyme family requires different metals, ligands, environment, etc., targeting a specific substrate molecule (or molecule family). As consequence, research of such enzyme mimetic ligand-metal complexes will probably not lose relevance for a long time.

General approaches

There are two distinctively different directions of metalloenzyme modeling, namely the structural and the functional enzyme model design. Model compounds are much easier to study than the whole native enzyme, and they can help gaining a better understanding over the structure-catalytic activity relationship. In structural enzyme modeling the main goal is to build molecular structures similar to the active site of the selected metalloenzyme, which can provide important spectroscopic and structural data relevant to the native active site. This can be achieved through e.g. comparison of smaller and larger peptide sequences responsible for metal binding, with respect to metal-ligand interactions or catalytic activity.

Functional enzyme models target most importantly the catalytic performance and its mechanism, not necessarily aiming for the active center-like structure. However, structural models often provide inspiration for functional enzyme model design, and the best enzyme mimetic complexes are engineered based on similarities to the active sites in e.g. substrate recognition, substrate activation or metal-metal interaction.

Structurally enhanced peptide compounds can serve as 'in-between' examples as enzyme models. Due to the amino acidic functions, the donor groups are identical to that of the metalloenzyme center, and other functional groups can facilitate substrate binding or acid/base catalysis, similarly to the native enzyme. Unlike linear peptide fragments, their structure is organized by a non-peptide-type scaffold, which may provide the rigidity necessary for a well-defined metal binding site. These structurally improved peptide derivatives can retain hydrolysis of the metal or the loss of the physiologically relevant binding mode, which are often the main sources of catalytic inefficiency in linear peptidemetal systems.

The literature can serve with countless examples on enzyme mimicking complexes. With respect to this work, three catalytic processes are presented below: oxidation of catechol, dismutation of superoxide radical anion and hydrolysis of activated phosphoesters.

Catechol oxidation

Catechol oxidases (COx) are monomeric enzymes with ~40 kDa molar mass. They contain a type-3 copper center with a Cu(II)-Cu(II) distance of approximately 2.9 Å in the native met form. Catechol oxidase enzyme was isolated by Krebs and co-workers in 1998 from sweet potato (Ipomoea batatas) [47], and its structural and catalytic analyses have been carried out. COx enzyme catalyzes the oxidation of ortho-diphenolic (catechol) compounds to the corresponding ortho-benzoquinones (Figure 1.6). The mechanism of the oxidation pathway was proposed by Eicken, Krebs and Sacchettini [49] (see Appendix A.1). During the catalytic cycle two substrate molecules are converted; one dioxygen molecule is consumed while two o-benzoquinone and two water molecules are released. Both copper(II) centers in the met form are surrounded by three His imidazole N atoms, along with a -hydroxo bridge between the two metal ions, altogether forming trigonal pyramidal coordination sphere per Cu(II). The bridging OH -group conducts a strong antiferromagnetic interaction between the copper centers, hence, the met form is EPR silent. After the oxidation of one substrate molecule, the deoxy state of the enzyme is formed with two Cu(I): their distance is significantly increased (~4 Å), one copper is in square planar geometry with an empty coordination site, the other remains in a distorted trigonal pyramidal structure with an additional water molecule. According to the suggested mechanism, oxygen uptake occurs through the formation of a peroxo - 2 : 2 intermediate, resulting the oxy form (Cu(II)-Cu(II) distance: 3.8 Å), which binds the second substrate molecule as well (Appendix A.1). Intramolecular electron transfer from the catechol to the peroxo-bridge results in the formation of the product and water, and the hydroxo-bridged oxidized form of the enzyme is recovered. The above described coordination modes of the copper centers and the fashion of the oxygen uptake is well preserved among other type-3 copper cores, as it was found in hemocyanin (Hc) or tyrosinase (Tyr) [50]. The conformation of the active center is only weakly influenced by the oxidation state of the metal ions, which emphasizes the importance of the robust, fixed three dimensional structure at the active site.

Catechol oxidation is an extensively used test reaction in enzyme mimicking studies, thus it has a large literature background. An often applied model substrate for catechol oxidase mimicking is the 3,5-di-tert-butylcatechol (H 2 DTBC), which is selectively oxidized to 3,5-di-tert-butyl-o-benzoquinone in presence of oxygen and copper(II) containing catalysts, without the cleavage of the aromatic ring. Such enzyme mimicking studies have been summarized in several review articles [51][52][53][54], presenting catalytic performance of mono-and multicopper(II) species, mechanistic considerations and substrate binding studies. Based on these informations, a few general observations can be established regarding catechol oxidase mimicking:

(1) Unlike the native enzyme, catechol oxidizing model complexes mostly release H 2 O 2 side-product instead of water, carrying out two electron reduction of dioxygen. There are a few examples where water formation is proposed [55,56] based on insufficient amount of H 2 O 2 formation detected; however, this finding can also be due to H 2 O 2 consumption along the catalytic oxidation, which indeed has been proven in some cases [56][57][58].

(2) The most active complexes were found among dinuclear copper(II) complexes, indicating that the close proximity of two copper centers is a necessity for highly efficient COx activity; however, numerous mononuclear copper(II) complexes also exhibit (lesser, but notable) catechol oxidase activity [54,[59][60][61][62][63][64][65].

(3) Anaerobic and aerobic substrate or substrate analog binding measurements revealed that binuclear complexes generally perform catechol oxidation through co-operation of metal centers (except binuclear complexes with distant copper(II) centers [56]) involving the formation of a -peroxo dicopper(II) intermediate, similarly to the native enzyme [57,[66][67][68][69][70][71][72][73]. Monocopper complexes, however, exhibit one electron oxidation of the substrate through the formation of a Cu(I)-semiquinonato adduct. This adduct reacts with O 2 leading to benzoquinone production.

(4) According to the one-electronic mechanistic pathway involving the formation of a semiquinone-type adduct, catalytic oxidation of catechol is also possible in absence of redox active metal ions: several zinc(II) complexes were found to show catechol oxidaselike activity [74][75][76][77][78], often in case of redox non-innocent (e.g. Schiff-base) ligand systems.

Copper(II) containing COx model systems have been summarized recently [54]. According to the Michaelis-Menten enzyme kinetic model, k cat (s -1 ) and K M (M) parameters represent the rate of product formation from the catalyst-substrate adduct and the stability of the latter adduct, respectively. Catalytic performance of enzyme mimics is often demonstrated by the k cat /K M ratio, conveniently representing the overall effectiveness of the catalyst. The most powerful catalyst to date has been synthesized by Das and co-workers [71], based on a Schiff-base ligand (see Figure 1.7. (a)); report on H 2 DTBC oxidase activity has been supplemented by substrate binding measurements. The k cat /K M value for the above-mentioned complex is 3913 M -1 s -1 , which is still two orders of magnitude lower than the value calculated for the native COx enzyme isolated from sweet potato [47]. Several other compounds exhibit outstanding catalytic activities in presence of copper(II); mostly symmetrical, N-donor ligands, and all of them provides the formation of dinuclear, Structures of ligand precursors of some highly active catecholase mimicking model complexes. Redrawn after (a) Das [71], (b) Meyer [67], (c) Klemm [69], (d) Casella [79], (e) Bouwman [68], (f) Reedijk [56] and co-workers.

Although these catecholase mimics are excellent functional models of the native catechol oxidase, there is hardly any resemblance in the nature of donor groups to the enzyme itself. In order to gain better understanding on the enzymatic reaction, and to design more high-performance catalysts in the future, catecholase mimicking capabilities of peptide-type ligand-metal complexes ought to be investigated.

Not many peptide-metal complexes have been studied so far for that purpose. Kinetic characterization of the copper(II)-Ac-His-His-Gly-His-OH tetrapeptide system has been carried out previously in our laboratory [65] using H 2 DTBC substrate; this study revealed that the {3N im ,OH -} coordinated CuH -1 L complex acts as efficient COx mimic in 86 wt% methanol-water medium (k cat = 0.063 s -1 ). Another investigated system is the copper(II)-Ac-His-Pro-His-His-NH 2 ; similarly, the 'extra' deprotonated CuH -1 L was assigned as the catalytically active complex, with a relatively high k cat = 0.12 s -1 , and with K M = 3.5 mM, implying moderate substrate binding. A few other peptide-copper(II) complexes were also shown to possess catalytic activity in oxidation of H 2 DTBC or catechol substrates [58,80,81], showing moderate k cat /K M values.

Aside of these separate examples, there are no comparative investigations or mechanistic arguments on the catecholase mimicking properties of copper(II)-peptide complexes in the literature. Therefore, their role and function as catechol oxidase mimics is to be further explored.

Superoxide dismutation

Superoxide dismutase (SOD) enzymes catalyze the disproportion of superoxide radical anion (O 2

-

) forming upon monovalent reduction of dioxygen. Cu,Zn-SOD is a homodimeric enzyme of 2×16 kDa present in eukaryotic organisms. Both domains contain a copper and a zinc ion in the active site, which are linked via a -imidazolato bridge ligand in the oxidized form. The remaining coordination sites of copper are occupied by three histidine side chains forming a distorted five-coordination geometry with a free coordination site, while the zinc(II) is surrounded by two imidazole rings and an aspartic -COO -group in a tetrahedral structure (Figure 1.8). Superoxide radical is bound by the Cu(II) in the active site; superoxide decomposition is achieved by the reaction with both the oxidized and reduced copper center as following:

Zn(Im -)Cu(II) + O 2 -  Zn(Im -)Cu(I) + O 2 Zn(Im -)Cu(I) + H +  Zn(ImH) + Cu(I) Cu(I) + O 2 -  Cu(II)-O 2
Superoxide dismutation is a frequently targeted subject in biochemical studies. The rate of the reaction is usually measured indirectly due to the high reactivity of O 2 -. One of the most often used test reaction for SOD activity determination is a modified version of the McCord-Fridovich method [83]: superoxide radicals are produced in situ by the xanthinexanthine oxidase system, and they subsequently react with the chromophoric nitrotetrazolium Blue choride (NBT), causing an increasing absorption band at 560 nm. Compounds with SOD activity inhibit this reaction by abstracting the superoxide reactant, therefore SOD performance of a complex can be given as IC 50 value (concentration of the complex where inhibition of the reaction NBT  NBT-diformazan is 50%). When comparing systems studied under different concentration conditions, SOD activity may be expressed as k, calculated as k NBT ×[NBT]/IC 50 [84], in order to obtain [NBT]-independent parameters. The scheme of the NBT model reaction is depicted on Figure 1.9. A huge variety of metal complexes has been investigated as antioxidants, including copper(II)-peptide SOD models. Table 1.2. presents IC 50 and k values of some investigated copper(II)-peptide complexes, along with that of free Cu 2+ (in phosphate buffer), and the native Cu,Zn-SOD as comparison. It appears that copper(II) complexes of histidine-containing peptides perform superoxide dismutation in a very effective manner; some of them can be considered as good structural models of the native enzyme, too. As it was mentioned before, the real challenge in the design of structural SOD models with peptides is the suppression of early amide nitrogen coordination. This was successfully achieved by Várnagy and co-workers, using sarcosine (N-methylglycine) in alternating positions with histidines; the CuL complex of {3N im } coordination forms in practically 100% at pH 7.4. The SOD activity of this species is only one order of magnitude lower than that of the native Cu,Zn-SOD [82].

Hydrolysis of phosphoesters

Hydrolase mimicking complexes are able to increase the rate of hydrolysis in activated phosphoesters by (1) providing the nucleophilic OH -group at relatively low pH, (2) by binding and activating the substrate, making it more vulnerable to nucleophilic attack and (3) destabilizing the binding of the product, forcing it to dissociate from the active site. Beside electronic effects, the orientation of the substrate by the metal center is also important: steric preferences can result advantageous specificity/selectivity for the catalytic complex. Phosphoester hydrolysis is frequently targeted as subject of catalytic studies (Figure 1.10), since the reaction can be considered as a model for RNA or DNA cleavage. The most commonly studied activated phosphoester substrates are the minimalist DNA models bis(4-nitrophenyl) phosphate (BNPP) and 4-nitrophenyl phosphate (NPP), and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) as a simplified model of RNA.

Many copper(II) and zinc(II) complexes are reported as efficient catalysts in phosphoester hydrolysis, along with lanthanide species. Dinuclear complexes might be especially adequate as hydrolase mimics, due to the dual activation of the substrate by the dinuclear core: -hydroxo/alkoxo species are often found to be excellent candidates [92][93][94][95]. Mononuclear complexes can also exhibit hydrolase function; in this case, coordinated OH -group(s) or free coordination site(s) facilitate substrate binding and activation.

AIMS

The overall goal of this work was to design, synthesize and characterize new tripodal peptide-type ligands and their complexes with biologically relevant transition metal ions. With the combination of the structuring effect of tripodal scaffolds and the advantageous coordination properties of histidine, a new class of ligands can be obtained with advanced metal binding capabilities, suitable for potential catalytic applications. The main points of interest arisen within the scope of this thesis can be categorized as following:

(1) We wished to confirm that tripodal platforms are capable of improving metal binding of peptides, peptide-type ligands, as expected. Tripodal ligands provide exceptional stability for their metal complexes due to their enhanced chelating effect. Their suitable modification can open a window towards new functionalities based on appropriate metal binding. Therefore, detailed pH potentiometric measurements were carried out in order to determine the stability of the forming metal complexes, comparing them with structurally relevant examples from the literature.

(2) We wanted to reveal the significance of the tripodal and the amino acid moiety, with respect to the structure of the forming species. Beside thermodynamic stability, structural characterization of the metal complexes were equally important: 1 H and 2D nuclear magnetic resonance (NMR) studies were used to assign the coordinating groups in Zn(II) complexes, while combined UV-visible (UV-Vis), circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy was applied to study the coordination properties of Cu(II) complexes.

(3) The effect of N-and C-terminus, asymmetric functionalization and terminal protection in the ligands was to be investigated. The varying number of functionalized 'legs' provides varying number of donor groups available to metal ions. This, along with symmetry, is a leading force in the formation of multinuclear complexes, self-assembly and other interesting coordination features.

(4) The forming metal complexes were to be tested in simple enzymatic model reactions, including oxidoreductase and hydrolase functions. Pseudopeptide ligands with enhanced stability are promising candidates as components of enzyme mimics; therefore all ligandmetal systems were tested as catalysts in oxidative and hydrolytic reactions. We were interested in kinetic description of the catalytic systems, as well as in mechanistic details (substrate binding, effects of reaction partners etc.).

(5) We meant to draw conclusions regarding the relation of the experienced catalytic behavior and the structure of the applied ligand. Catalytically active peptide-metal complexes have great relevance from the point of view of structural enzyme modeling. Studying several tripodal peptides, we wanted to learn more about the development of catalytic performance of peptide complexes. Such findings, connecting structural properties and activity, can be powerful tools in future enzyme mimic design.

MATERIALS AND METHODS

SYNTHESIS OF THE LIGANDS

Preparation and characterization of the investigated ligands has been performed in our laboratories, at the University of Szeged. The quality and purity of all synthesized materials has been verified by ESI-MS, 1 H and 13 C NMR methods (see Appendix A.2-A.20). Starting materials were all analytically pure reagents and were used without further purification.

Synthesis of the N-terminal tris-His-ligand: tren3his (L 1 )

Pre-treatment of Boc-His(Boc)-OH. Boc-His(Boc)-OH starting material was obtained from acidic treatment of Boc-His(Boc)-OH dicyclohexylammonium (DCHA) salt precursor (Sigma-Aldrich) according to the following procedure: 3 g Boc-His(Boc)-OH . DCHA (5.59 mmol) was suspended in 100 ml ethyl acetate, washed with 30 ml ice cold phosphorous acid solution (10 (m/m)%) and cold water until neutral pH. The acidic phase was washed with 40 ml fresh cold ethyl acetate. The organic phases were combined, free Boc-His(Boc)-OH was obtained after evaporation of ethyl acetate solvent.

Synthesis of L 1 . 1.38 g Boc-His(Boc)-OH (3.88 mmol), 0.225 g tren (1.54 mmol, Sigma-Aldrich), 0.55 g HOBt (4.07 mmol, NovaBiochem) and 0.83 g DCC (4.02 mmol, Sigma-Aldrich) was dissolved in 40 ml dichloromethane with stirring. The reaction was followed by thin-layer chromatography (eluent: 10% MeOH-90% chloroform +3% DIPEA). After 3 h at room temperature the white precipitate (dicyclohexylurea) was removed by filtration and dichloromethane was evaporated from the remaining solution. The residue was treated with a mixture of 95 (V/V)% trifluoroacetic acid (TFA), 2.5 (V/V)% triisopropylsilane (TIS) and 2.5 (V/V)% distilled water in order to remove N-protecting Boc-groups. TFA was evaporated using liquid nitrogen trap, the oily product was dissolved in water, neutralized with NaOH, filtered and purified by preparative HPLC (reverse-phase (RP) column: Supelco C18 5 m, 25 cm × 10 mm geometry, 0 to 4.1% MeCN +0.05% TFA/20 min. gradient elution mode, 3 ml/min flow rate, t R ' = 8.6 min). After lyophilization the trifluoroacetate salt of L 1 was obtained as white hygroscopic solid (61% yield). 1

Synthesis of the N-terminal asymmetric His-ligands: tren2his (L 3 ), tren1his (L 4 )

Preparation of the asymmetric trenXhis ligands was realized in two steps, since the partial protection of tren had to be accomplished first, in order to carry out an effective asymmetric peptide coupling in solution phase.

Synthesis of mono-Boc tren.

Mono-Boc protected tren was necessary for the selective synthesis of tren2his. 25.5 ml tren (175 mmol) was dissolved in 150 ml dioxane. To that solution, 6.0 g di-tert-butyl dicarbonate (Boc 2 O, 27.5 mmol) in 150 ml dioxane was added dropwise applying N 2 atmosphere. After 17 h of stirring at r.t., a white precipitate was formed. Dioxane was evaporated, the white solid was dissolved in water. The aqueous solution was extracted by 6×75 ml dichloromethane. Organic phases were combined, the evaporation of DCM solvent resulted the mono-Boc protected tren as pale yellow oily product of high viscosity. Purity was checked by thin-layer chromatography (eluent: 75% MeCN-15% H 2 O-10% ccNH 3 solution) and NMR. 1 Synthesis of di-Boc tren. Di-Boc protected tren, as a starting material for tren1his was synthesized following the description below: 2.34 g tren (16.0 mmol) was dissolved in 800 ml dry MeOH, to that a solution of 5.94 g Boc 2 O (27.2 mmol) in 140 ml dry MeOH was added slowly, dropwise, at 0 • C. The mixture was put on ice for two days, enabling a slow reaction between the components. After the reaction time, thin-layer chromatograms indicated the presence of free, mono-, di-and tri-Boc tren accordingly. MeOH was evaporated, the residue was dissolved in chloroform, and the mixture was washed with four portions of 10% NaHCO 3 solution, removing mono-Boc and free tren from the organic phase. Di-Boc tren was obtained after chromatography on silica normal phase column (eluent: 50% MeOH-50% chloroform +2% ccNH 3 solution) and evaporation, as a pale yellow oily product. The purity of the compound was verified by NMR. 1 H NMR (500 MHz, CDCl 3 )  (ppm): 1.36 (s, 18H), 2.54 (br t, 4H), 2.65 (br t, 2H), 3.09 (br t, 6H).

Synthesis of Boc-His(Boc)-OH. Boc-His(Boc)-OH starting material was also prepared for these syntheses based on a general literature procedure [96], as following: 1.8 g L-His (11.6 mmol, Sigma-Aldrich) was suspended in 80 ml 50% tetrahydrofurane/water mixture. 3 g NaHCO 3 (35.7 mmol) and 6 g Boc 2 O (27.5 mmol) was added at 0 • C. The turbid reaction mixture was stirred for 30 min at this temperature, then for further 14 h at r.t., becoming a clear solution. The mixture was washed with diethyl ether (2×50 ml), followed by careful addition of ~7 M solution of citric acid until pH reached 4.5. The aqueous solution was extracted with DCM (3×50 ml), the organic phases were combined and the solvent was evaporated. Boc-His(Boc)-OH was obtained as sticky white solid (31% yield), purity was confirmed by HPLC, by comparison with a standard Boc-His(Boc)-OH sample (RP column description in section 3.1.1. Eluent: isocratic 65% MeCN-35% water, t R = 9.1 min).

Synthesis of L 3 . 0.84 g Boc-His(Boc)-OH (2.36 mmol) and 0.27 g mono-Boc tren (1.10 mmol) were dissolved together in 50 ml dichloromethane, 0.47 g DCC (2.30 mmol) and 0.31 g HOBt (2.30 mmol) were added subsequently. The mixture was stirred overnight at rt. After filtration and evaporation of the solvent, the cleavage of Boc protecting groups was accomplished by a treatment of 95%-2.5%-2.5% TFA/TIS/water mixture. The cleaving mixture was evaporated using liquid nitrogen trap, the oily residue was dissolved in water, neutralized and purified by preparative HPLC (RP column description in section 3.1.1., eluent: 0 to 3.2% MeCN +0.1% TFA/20 min gradient elution mode, 3 ml/min flow rate, t R ' = 9.2 min). After lyophilization the trifluoroacetate salt of L 3 (62.7% yield) was obtained as white solid. 1 Synthesis of L 4 . 0.42 g Boc-His(Boc)-OH (1.18 mmol) and 0.38 g di-Boc tren (1.09 mmol) were dissolved together in 50 ml dichloromethane, 0.25 g DCC (1.20 mmol) and 0.16 g HOBt (1.20 mmol) were added subsequently. The mixture was stirred overnight at r.t. After filtration and evaporation of the solvent, the cleavage of Boc protecting groups was accomplished by a treatment of 95%-2.5%-2.5% TFA/TIS/water mixture. The cleaving mixture was evaporated using liquid nitrogen trap, the oily residue was dissolved in water, neutralized and purified by preparative HPLC (RP column description in section 3.1.1., eluent: 0 to 2% MeCN +0.1% TFA/15 min gradient elution mode, 3 ml/min flow rate, t R ' = 3.7 min). After lyophilization the trifluoroacetate salt of L 4 (70.6% yield) was obtained as white solid. 1

Synthesis of the C-terminal tris-His-ligand: nta3his (L 2 )

General issues. The preparation and purification of the C-terminal nta3his was found to be rather challenging. The tripodal scaffold molecule nta has a poor solubility in most of the common solvents, especially those being suitable media for peptide coupling in solution phase (e.g. DCM, chloroform). For that reason, reaction time became undesirably long, in respect of the relatively low stability of the coupling agents used in the procedures. In addition, as a C-terminally protected His precursor was necessary for the synthesis, the commercially available L-His-methylester . 2HCl was applied. This raised the need of using a base, in order to deprotonate the N-terminus of the amino acid and thus generating the nucleophilic group. The usage of bases in the carbodiimide coupling strategy, however, was proved to inhibit the activation of the carboxylic function [97], slowing down the reaction even further. The application of the commonly used organic base diisopropylethylamine (DIPEA) caused additional problems, as it was impossible to find an appropriate gradient program to separate it entirely from the product: yet HPLC chromatograms indicated a single peak, traces of DIPEA were found in the sample of purified nta3his by 1 H NMR and ESI-MS as well. To avoid this, triethylamine (TEA) was used instead as base in small quantities during the following syntheses, resulting a product of eligible purity. However, it must be remarked that despite the various attempts for optimization, the yield of the reaction remained considerably low, the purification process was extremely long and the amount of obtained product was very limited. Synthesis of L 2 . 1 g L-His-methyl ester . 2HCl (4.13 mmol, Sigma-Aldrich) and 0.852 g DCC (4.13 mmol), 0.558 g HOBt (4.13 mmol) coupling agents were dissolved in 60 ml chloroform in presence of triethylamine (1.2 ml). A suspension of nitrilotriacetic acid (0.26 g, 1.36 mmol) was added to the mixture and stirred at room temperature overnight. Chloroform was evaporated, the oily residue was treated with 5 eq. NaOH solution (H 2 O:MeOH 2:1), in order to remove the C-terminal protecting group. The hydrolysis of methyl ester group was completed in 17 h at r.t. Excess base was neutralized, the mixture was filtered and purified by preparative HPLC (RP column described in section 3.1.1, eluent: 0 to 5% MeCN +0.1% TFA/20 min. gradient elution mode, 3 ml/min flow rate, t R ' = 14.8 min). After purification and lyophilization, L 2 was obtained as a trifluoroacetate salt (yield 53%). 1

Syntheses attempted on solid phases: nta1his and nta1his-NH 2

The encountered difficulties regarding the synthetic strategy of C-terminal His compounds forced us to open towards different synthetic procedures. Beside the optimizations of solution phase peptide coupling, solid phase synthetic methods were also experimented with. Attempts for such kind of preparations for tripodal ligands were already reported in the literature [26,98]; it is a convenient method for synthesizing partially functionalized compounds, since small tripodal scaffold molecules are being attached by only one 'arm' to the resin. Therefore we applied the Fmoc-strategy for creating the mono-His functionalized, C-terminally free and protected nta1his and nta1his-NH 2 ligands, respectively.

Although early 1 H NMR and ESI-MS measurements indicated unified, pure products following the syntheses, the materials turned out to be degrading slowly, even after repurification. This eventual degradation could not be prevented even by careful storage in solid state, at -4 • C. Therefore, experimental data regarding these compounds are limited.

Synthesis of nta1his.

The mono-functionalized C-terminal nta1his was prepared using Wang resin (100-200 mesh, Merck). 1 g of resin was left to swell together with 2 eq. Fmoc-His(Trt)-OH (Sigma-Aldrich) in DMF for 15 min. 3.3 eq. pyridine (Sigma-Aldrich) and 2 eq. 2,6-dichlorobenzoyl chloride (Molar Chemicals) was added and stirred overnight. DMF was filtered, the resin was rinsed with N-methyl-2-pirrolidone (NMP), as in every following rinsing step. Remaining active sites on the Wang resin have been blocked by treatment with 4 eq. pyridine/benzoyl chloride (Molar Chemicals) mixture. Fmoc group of His was removed by 20% piperidine solution, the presence of the free NH 2 groups were confirmed by Kaiser-test [99]. Nta (4 eq.) was coupled then to the amino functions using HOBt (4 eq., Novabiochem), HBTU (4 eq., Novabiochem) and DIPEA, stirred together for 4 h. The coupling reaction was repeated, negative Kaiser-test attested for the absence of residual free amino groups. The ligand was cleaved from the resin using a mixture of 95%-2.5%-2.5% TFA/TIS/water. After the evaporation of TFA, the residue was dissolved in water, neutralized and purified by preparative HPLC (RP column described in section 3.1.1, eluent: 100% H 2 O+0.1% TFA/10 min. isocratic elution mode, 3 ml/min flow rate, t R ' = 2.2 min). After lyophilization the trifluoroacetate salt of nta1his was obtained as white hygroscopic solid. 1

Synthesis of nta1his-NH 2 .

The C-terminally protected compound was synthesized similarly to nta1His, except for the followings: Rink Amide AM resin (0.68 mmol/g) was used for the preparation, to which Fmoc-His(Trt)-OH was coupled straightforwardly after the removal of Fmoc protecting group, using 4 eq. HOBt, 4 eq. HBTU coupling agents in presence of 8 eq. DIPEA. The coupling step was repeated, after 45 min stirring Kaiser-test supported the absence of residual free amino groups. Fmoc group of His moiety was removed and the coupling of nta followed as described previously. HPLC purification (RP column described in section 3.1.1, eluent: 100% H 2 O+0.1% TFA/10 min. isocratic elution mode, 3 ml/min flow rate, t R ' = 2.7 min) and lyophilization resulted nta1his-NH 2 trifluoroacetate salt as white solid. 1

CHARACTERIZATION TECHNIQUES OF SOLUTION EQUILIBRIUM AND STRUCTURE

pH potentiometry

The protonation and coordination equilibria were investigated by pH-potentiometric titrations in aqueous solutions (I = 0.1 M NaCl and T = 298.0 ± 0.1 K) under argon atmosphere. The titrations were performed using a PC controlled Dosimat 665 (Metrohm) automatic burette and an Orion 710A precision digital pH-meter. The Metrohm micro glass electrode (125 mm) was calibrated by the titration of hydrochloric acid and the data were fitted using the modified Nernst-equation [START_REF] Rosotti | The determination of stability constants[END_REF]:

] H [ ] H [ ] H log[ w OH H 0           K J J K E E
where J H and J OH are the fitting parameters representing the acidic and alkaline error of the glass electrode, respectively (K w = 10 -13.75 is the autoprotolysis constant of water [START_REF] Högfeldt | Stability Constants of Metal-Ion Complexes, Part A. Inorganic Ligands[END_REF]). The parameters were calculated by the non-linear least squares method. The formation of the complexes was characterized by the general equilibrium process:

pM + qH + rL M p H q L r M p H q L r  r q p r q p L H M ] L [ ] H [ ] M [ ] L H M [ r q p  
where M denotes the metal ion and L the fully deprotonated ligand molecule. Charges are omitted for simplicity. The protonation constants of the ligands and the formation constants of the complexes were calculated by the PSEQUAD computer program [START_REF] Zékány | PSEQUAD for chemical equilibria[END_REF] from 4, and 6-8 independent titrations respectively (ca. 90 data points per titration). The metal ion concentrations varied between 0.5-3.3×10 -3 M, using 2:1, 3:2, 1:1 and 1:2 metalto-ligand ratios.

UV-visible (UV-Vis) spectroscopy

In order to gather information about the number and quality of the coordinating groups around the metal ion(s), UV-Vis spectrometric measurements were performed. Changes in the coordination sphere can be monitored by the appropriate interpretation of pHdependent UV-Vis spectra series.

Light absorption of a complex can be originated from either the ligand (chromophoric groups, n*, *, n* transitions), the metal (d-d transitions) or both (M-L or L-M type charge transfer (CT) bands). Newly appearing absorption bands or displacements imply a change in coordination environment around the metal ion. However, qualitative interpretation of spectral changes is not always simple, since the d-d transitions and HOMO-LUMO energy gap is dependent of many factors, such as the quality of the donor groups, the coordination geometry, symmetry and distortions of the coordination spheres, temperature, solvent, etc.

Regarding metal complexes with relatively simple donor groups, there has been an effort in the literature to state some useful empirical 'rules', guidelines for treating and comparing the behavior of different metal-peptide systems. The most important of these works concerned the relation between the energy of the d-d band of Cu(II), expressed as wavenumbers (/cm -1 ) and the quality/number of donor groups in the equatorial plane. This was first published by Billo and co-workers [START_REF] Billo | [END_REF] and later extended by Sigel and Martin [104], Pettit [START_REF] Pettit | Perspectives on Bioinorganic Chemistry[END_REF] and Prenesti [START_REF] Prenesti | [END_REF], who afterwards continued researching on the effects of axially coordinating donor groups to the visible spectra of Cu(II) complexes as well, estimating their contribution to the  max [107].

Optical spectroscopic methods such as UV-Vis and CD spectroscopy enable the possibility of quantitative characterization: the Beer-Lambert law defines the relationship between the concentration of the absorbing species (c) and the measured absorbance (A) according to the following equation:

A = lg(I 0 /I) = ×c×l,
where I 0 denotes the intensity of the incoming light, I the intensity of the transmitted light,  is the molar extinction coefficient (M -1 cm -1 ) of the absorbing species and l (cm) is the optical pathlength.

UV-Vis spectra were recorded on a Unicam Helios  spectrophotometer, a MOS 450 (BioLogic) spectrometer and a PerkinElmer Lambda 1050 spectrophotometer, using a quartz cuvette with 1 cm optical pathlength. Spectral changes were monitored between pH 2 and 11 for all Cu(II)-ligand systems, at all relevant Cu(II)-to-ligand ratios. The metal ion concentration varied between 8.8×10 -4 -2.4×10 -3 M. The pH-dependent UV-Vis spectra were processed together with the pH-potentiometric data using the PSEQUAD computer program [START_REF] Zékány | PSEQUAD for chemical equilibria[END_REF], resulting logvalues as well as individual UV-Vis spectra of the Cu(II) complexes.

Circular dichroism (CD) spectroscopy

Similar qualitative and quantitative information can be extracted from CD spectra. CD spectroscopy uses plane polarized light which can be split to left-and right-handed circularly polarized components. Optically active molecules absorb different quantities of these components, this phenomenon is called circular dichroism. In CD spectroscopy we obtain a spectrum by the difference of extinction coefficients of the two components (Δε = ε left -ε right ) at each scanned wavelength (). The Beer-Lambert law is applicable for the qualitative treatment of the data, enabling to calculate individual CD spectra for light absorbing chiral metal complexes.

Chirality of transition-metal complexes can be assigned to the asymmetry sources summarized below:

(1) Central chirality, which corresponds to the chiral distribution of donor groups around the metal ion,

(2) Helical chirality, which is caused by the chirally positioned chelate rings around the metal center,

(3) Conformational chirality of the chelate rings (4) Chirality of the coordinating ligand, which is conducted towards the metal ion through chemical bonds.

While effects (1)-( 3) have significance in case of kinetically inert complexes (mainly Co(III), Cr(III), Rh(III), Ir(III) and Pt(IV) complexes), chirality of copper(II) containing complexes are usually originated from the chiral ligand. In a CD-active complex, at least one of the conformers of the forming chelate ring must be stabilized, otherwise racemization would occur, resulting zero overall signal on the CD spectrum. The shape of a CD spectrum is unique of the three-dimensional environment of the central metal ion.

CD spectra were measured on a Jasco J-710 spectropolarimeter and a MOS 450 (BioLogic) spectrometer, in the wavelength interval from 300 to 900/830 nm, in a cell with 1 cm optical pathlength. Spectral changes were monitored between pH 2 and 11 for all Cu(II)-ligand systems, at all relevant Cu(II)-to-ligand ratios. The metal ion concentration varied between 8.8×10 -4 -2.4×10 -3 M. pH-dependent CD spectra were processed together with the pH-potentiometric data using the PSEQUAD computer program [START_REF] Zékány | PSEQUAD for chemical equilibria[END_REF], resulting logvalues as well as individual CD spectra of the Cu(II) complexes.

Electron Paramagnetic Resonance (EPR) spectroscopy

EPR spectroscopy can be used to structural characterization of systems bearing unpaired electron(s) such as radicals or paramagnetic metal complexes. The method is based on the Zeeman effect, namely that an external magnetic field splits the energy levels of an unpaired electron. This energy separation corresponds to the energy of microwave frequency.

EPR signals are dependent of the interactions between the unpaired electron and all non-zero nuclear spin(s) or other unpaired electron(s). The main interactions which must be taken into account in complex systems, are the following:

(1) Interaction of unpaired electron(s) of the metal ion with its non-zero nuclear spin (I≠0): EPR signal is split into a number of 2I+1 components. This interaction is characterized by the hyperfine coupling constant (A).

(2) Interaction of the unpaired electron with a non-zero nuclear spin of a different atom (donoratom of the ligand): EPR signal can be split into a number of 2nI+1 components. This interaction is characterized by the super hiperfine coupling constant (A).

(3) Dinuclear complexes can produce antiferromagnetic interaction between the metal centers, which denotes the interchange of electron spins. This interaction is highly sensitive to the proximity of the interacting metals, and can cause extreme broadening or quenching of the EPR signal.

EPR spectra of the copper(II) complexes were recorded on a BRUKER EleXsys E500 spectrometer (microwave frequency 9.81 GHz, microwave power 10 mW, modulation amplitude 5 G, modulation frequency 100 kHz), or a continuous wave Bruker EMXplus 10/12 spectrometer (microwave frequency 9.412 GHz). Since common copper(II) perchlorate was used for all measurements, the spectra were calculated by the summation of spectra 63 Cu and 65 Cu weighted by their natural abundances. The hyperfine and the relaxation parameters were obtained in field units (Gauss = 10 -4 T).

We applied EPR titrations in case of Cu(II)-L 1 : pH-dependent (pH = 2.3-11.3) series of EPR spectra were collected under argon atmosphere at room temperature, in both equimolar solution (0.95× [L 1 ] tot = [Cu(II)] tot = 1.6×10 -3 M) and at metal ion excess (1.40× [L 1 ] tot = [Cu(II)] tot = 2.31×10 -3 M) in a circulating system using a Heidolph Pumpdrive 5101 peristaltic pump. The ionic strength was adjusted to 0.1 M with NaCl. The two series of room-temperature CW-EPR spectra were simulated by the 2D_EPR program [108]. The parameters g o , A o Cu copper hyperfine (I Cu = 3/2) and A o N nitrogen (I N = 1) hyperfine coupling has been taken into account to describe each component curve.

The relaxation parameters, , , and  defined the linewidths through the equation

 MI =  + M I + M I 2
, where M I denotes the magnetic quantum number of copper(II) ion.

EPR spectra of Cu(II)-L 2 , -L 3 and -L 4 systems have been measured using individually prepared and pH-adjusted solutions (0.1 M NaCl ionic strength) in a capillary, at 1:1 and 2:1 metal-to-ligand ratios ([Cu(II)] tot = 0.8×10 -4 -3.3×10 -3 M). Samples were prepared as aqueous solutions; 100 l volumes were put in capillary, 20 l methanol was added to each sample in order to avoid water crystallization. The frozen solution EPR spectra were simulated by the "EPR" program [109], rhombic g-tensor (g x , g y , g z ), copper hyperfine tensor (A x , A y , A z ) and the orientation dependent linewidth parameters were fitted to set up the spectra. In cases when well resolved superhyperfine lines were detected, a rhombic nitrogen coupling was taken into account (a x N , a y N , a z N ) as well. The linewidths have been described by the above-mentioned equation using orientation dependent ,  and parameters. Isotropic EPR spectra, recorded at room temperature, could only be obtained for Cu(II)-L 2 complexes; they were fitted by the "EPR" program [109] using the parameters: g o , A o Cu copper hyperfine (I Cu = 3/2) and A o N nitrogen (I N = 1) hyperfine couplings. Detection of the oxygenated radical intermediate in Cu(II)-L 1 -H 2 DTBC system was carried out using a Bruker ECS106 X-band EPR spectrometer (modulation amplitude 0.84 G, microwave power 5 mW). After mixing the reaction solution under aerobic conditions (in EtOH/H 2 O 50/50 HEPES/CHES buffer,

I = 0.1 M, [complex] tot = 5×10 -5 M, [H 2 DTBC] = 2×10 -3 M, pH = 8
), a sample of 4-5 l was put in a capillary tube, and measured in presence of 2,2-diphenyl-1-picrylhydrazyl (DPPH) internal reference.

Nuclear Magnetic Resonance (NMR) spectroscopy

One of the most powerful structural characterization techniques is the NMR spectroscopy. It can be applied to species possessing non-zero nuclear spin (I ≠ 0), and is based on the fact that external magnetic field induces the splitting of the energy levels of these nuclear spins (number of energy levels is 2I+1). Resonance between the split energy levels can be achieved by radiofrequency irradiation. This frequency (also called Larmor frequency) is unique for each nuclear spin and is dependent of the external magnetic field.

The high number of signals on a simple NMR spectrum is due to the sensitivity of the resonance frequency to the chemical environment of a nuclear spin. This effect is called the chemical shift (), usually given in Hz or ppm, and it is in strict relation with the electronic shielding of the nuclei. Generally, the intensity of the signals is corresponding to the number of nuclei the signals are originated from, which contributes to the important structural information. Moreover, the spins are sensitive to the chemical environment of the neighboring atoms, resulting characteristic splitting (nI+1) of each signals. The interaction between the neighboring nuclei and therefore the induced splitting of the signals can be described by the coupling constant (J) in Hz.

Although NMR spectroscopy provides various and detailed structural information as it is, investigation of more complicated systems (e.g. proteins, multicomponent mixtures) often requires special techniques providing complementary data. 2D NMR techniques are based on transfer of magnetism between different groups, which results additional information about the interactions between further (not neighboring) functional groups via chemical bonds (e.g. COSY = correlation spectroscopy, TOCSY = total correlation spectroscopy) or space (ROESY = rotating-frame Overhauser spectroscopy). It is possible to execute resonance transfer between nuclei of different elements (HMQC = heteronuclear multiple-quantum correlation, HMBC = heteronuclear multiple bond correlation), and thus to assign every peak of the 1D spectra to the corresponding groups. In a system prone to aggregation, the measurement of diffusion processes can be extremely helpful in order to estimate the size of species occurring in the solution (DOSY = diffusionordered spectroscopy), which can be achieved by monitoring the different relaxation times of the species with different size.

The width of NMR signals depends on many factors; some of them are practical (frequency of the instrument, homogeneity of the magnetic field, etc.). However, in case of diamagnetic metal complexes, the kinetics of complex formation has major importance on the matter. Basically, the relation between the rate of complex formation and the relaxation time of the studied nucleus determines the width of the signal.

(I) In case the ligand exchange rate around the metal is considerably faster than the relaxation time of the observed nuclei, the obtained peak will be in fact a superposition of many different states (conformation, chemical environment) regarding the nucleus. Signals are usually narrow, free and complexed ligand peaks can not be differentiated.

(II) In case the ligand exchange rate is much slower than the relaxation of the studied nuclei, the obtained spectra will represent a single state corresponding to the metal complex. Signals are very sharp and multiplication of them often occurs, indicating chemically different environments (for each complexes and free ligand).

(III) If the ligand exchange rate is very close (in the same order of magnitude) to the rate of relaxation, signals are broadened. It is considered an intermediate exchange rate in respect of the NMR time scale and usually indicates some sort of interaction, which causes the loss of free motion of the molecule part involved, and therefore a retarded exchange rate.

(IV) Presence of paramagnetic compounds (e.g. paramagnetic transition metal ions) causes significant changes on the NMR signals. In presence of copper(II), due to its slow electronic relaxations, important broadening of the signals can be observed. Although, some information (like J-couplings) might be lost by the decreased resolution of the spectra, the selective broadening may help to identify the binding site(s) of metal ions.

NMR spectra were recorded in Szeged (Hungary), on a Bruker Avance DRX500 spectrometer (500.13 MHz frequency), and in Nancy (France), on a Bruker DRX400 spectrometer (400 MHz frequency). All samples were aqueous solutions with 10-20% D 2 O unless it is marked otherwise. Water signal suppression on 1 H spectra has been achieved using presat or watergate pulse sequences. Dioxane was used at internal reference ( dioxane = 3.70 ( 1 H) and 66.5 ( 13 C) ppm). Ligand concentrations have been adjusted to 1-2 mM unless stated otherwise. The assignment of the proton and carbon signals of the ligands was based on 1 H-1 H COSY, 1 H- 13 C HMQC and 1 H- 13 C HMBC 2D spectra (see Appendix A.9-A.20). DOSY measurements of Zn(II)-L 1 3:2 system were performed at the University of Debrecen, with solvent suppression, using 64 gradient step at 293 K. Three sets of spectra were recorded, following the preparation of the complex solution and after 90 and 156 h, in order to verify the kinetic stability and reproducibility of the system during the experiment. Data were processed using the Topspin 2.0 software package (Bruker).

Mass Spectrometry (MS)

Mass spectrometric measurements were carried out in order to verify the presence our synthesized ligand molecules and their metal complexes forming at various metal-to-ligand ratios. In most cases, free ligand molecules and monocomplexes were analyzed using electrospray ionization (ESI) quadrupole MS coupled to a liquid chromatography column. Di-or trinuclear complexes were detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS method, either in the Biological Research Centre of Szeged (Hungary), or in the University of Lorraine (Metz, France). The samples were prepared in aqueous solutions at the desired metal-to-ligand ratios, pH was adjusted using NaOH and HCl solutions. ESI-MS measurements were carried out by direct injection of the samples to the LC column. In case of MALDI measurements, 1 µl of the samples was spotted onto the target plate and let dry before adding 0.5 µl of the matrix solution (20 mg/ml 2,5-dihydroxybenzoic acid (DHB) in water or 2,6-dihydroxyacetophenone (DHAP) in MeCN-water 1:1). Mass spectra were recorded in the positive (L 1 , L 3 and L 4 complexes) and negative (L 2 , nta1his-NH 2 complexes) reflectron modes collecting multiple shots for each sample (m/z range: 900-2000). External calibration affording ±200 or 100 ppm mass accuracy was performed using peptide standards, or polyethylene glycol standard (Aldrich). DHB and DHAP matrices were obtained from Sigma-Aldrich.

The mass spectrometers used in this work are the following: Finnigan TSQ 7000 ESI-LC-MS with triple quadrupole analyzer (University of Szeged, Department of Medical Chemistry, Szeged), Bruker MicroTOFq high-resolution ESI-MS (Université de Lorraine SRSMC UMR 7565, Nancy), Bruker REFLEX III MALDI-TOF MS (Biological Research Centre, Szeged), Bruker REFLEX IV MALDI-TOF MS (Université de Lorraine LCP-A2MC, Metz).

KINETIC EXPERIMENTS

In order to characterize the potential catalytic abilities of our complexes, kinetic experiments have been carried out, testing redox (SOD, catechol oxidation) and hydrolytic functions (phosphoester hydrolysis) as well. In all cases we used spectrophotometric detection of the forming product, resulting absorbance vs. time data series. All kinetic experiments were executed and treated according to the initial reaction rates method.

The initial reaction rates method presumes that the conversion of the substrate does not exceed 3-5%, where the quantity of product increases linearly in function of time. Initial reaction rate (v i ) can be calculated from the slope of this region of the absorbance-time curve:

dA/dt = ε λ ld[P]/dt

v i = d[P]/dt = -d[S]/dt = (dA/dt)/(ε λ l)
where dA/dt is the observed time-dependent absorbance (slope), [P] is the concentration of the product, [S] is the concentration of the substrate,   is the molar extinction coefficient of the forming product at the absorption maximum and l is the optical pathlength in cm.

Under pseudo-first order conditions, a pseudo-first order reaction rate constant (k obs ) can be introduced. Presentation of kinetic data usually includes the second order rate constant k', which is independent of the concentration of the catalyst, therefore can be used for correlating data from different concentration conditions.

v i = d[P]/dt = k obs [S] = k'[catalyst][S]
The Michaelis-Menten kinetic method was originally introduced to enzymatic reactions. In such reactions (considering only the most simple case as model) the enzyme (E) and the substrate (S) molecules form an adduct (ES) in a pre-equilibrium step, followed by the conversion of the substrate to product (P) with the regeneration of the enzyme.

E + S k a k' a ES k b P + E
Presuming steady-state approximation to the formation and consumption of ES, product formation can be expressed in differential equations as following:

, ] [ ] [ o E k dt P d  ] [ ] [ S K S k k M b  
, where [E] o is the total concentration of the enzyme and K M is the Michaelis constant:

a a b M k k k K '   .
In order to apply this methodology in our complex catalyzed reactions (where the complex = E), the following conditions must be settled: (I) Substrate concentration exceeds considerably the enzyme concentration. (II) The variation of substrate concentration is negligible during the reaction (which meets the prerequisites of the initial reaction rates method). (III) Product does not form a stable adduct with the enzyme. (IV) One substrate molecule binds to the enzyme.

Michaelis-Menten mechanism states a linear correlation between the reaction rate and [E] o , but the substrate-dependence is more complex: if [S]>>K M , then the reaction rate becomes independent on [S]:

d[P]/dt = k b [E] o If [S]<<K M ,

the reaction rate increases linearly with [S]

:

d[P]/dt = (k b /K M )[E] o [S]
According to these boundary statements, the experimental substrate-dependent reaction rate curve is a saturation curve, which can be used to calculate K M and k b (often cited as k cat ) parameters by e.g. least squares methods.

Catechol oxidation measurements

Kinetics. The oxidation of 3,5-di-tert-butylcatechol (H 2 DTBC) was followed in buffered solutions (0.02 M HEPES/CHES, I = 0.1 M (0.08 M NaCl), T = 298 K) by detecting the increase in absorption at 400 nm of 3,5-di-tert-butyl-o-benzoquinone (DTBQ, ε = 1900 M -1 cm -1 ). 50% EtOH-50% aqueous solutions were used to prevent the precipitation of DTBQ. The actual pH (used as pH corr ) in this mixed solvent was determined by substracting 0.21 units from the pH-meter reading [110], the auto-ionization constant of water in this medium is pK w = 14.84 [111]. Precise concentrations of O 2 were obtained by addition of EtOH saturated by O 2 gas to the reaction solutions (the solubility of O 2 is 250 mgdm -3 and 41 mgdm -3 in ethanol and water, respectively [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF]). After mixing the ethanol and the aqueous phases, the solutions were thermostated at 298 K for 2-3 minutes, then the reaction was initiated by the addition of H 2 DTBC as ethanolic solution.

Autooxidation of H 2 DTBC was taken into account in every experiments, resulting k obs,corr values (k obs,corr = k obs -k obs,auto ). The parameters of the Michaelis-Menten model (K M and k cat ) were calculated by non-linear least square method. All kinetic data are averages of at least three parallel measurements.

Substrate binding. Substrate binding experiments were carried out in buffered 50%

EtOH-H 2 O solutions (0.05 M HEPES/CHES, I = 0.1 M (0.05 M NaCl), T = 298 K), using 4-nitrocatechol (H 2 4NC) and H 2 DTBC. During the study of H 2 4NC binding the complex concentrations varied between 0.8 -2.5×10 -4 M or was held constant on 2.1×10 -5 M. Deprotonation constants and spectroscopic parameters of 4NC species were calculated using PSEQUAD program [START_REF] Zékány | PSEQUAD for chemical equilibria[END_REF].

Anaerobic H 2 DTBC binding UV-Vis measurements were executed under argon atmosphere, in closed quartz cuvette equipped with a septum, with 1 cm optical pathlength. Different volumes of H 2 DTBC stock solution was added to deoxygenized, buffered complex solutions using a Hamilton syringe. Complex concentrations were adjusted to 2.4×10 -4 M in case of each ligand system, substrate concentration varied between 0-1.2×10 -2 M. 'Blank' experiments, in the absence of metal complexes, were also carried out. Each measurement was performed twice in order to confirm reproducibility.

Detection of H 2 O 2 .

The formation of hydrogen peroxide during the oxidation of H 2 DTBC was identified and quantified spectrophotometrically, as [Ti(O 2 )(OH)] + complex ( 408nm = 935 M -1 cm -1 ), in aqueous solutions. In a typical experiment, 20 ml reaction solution has been prepared (0.02 M HEPES/CHES, I = 0.1 M (0.08 M NaCl), T = 298 K); 4.5 ml aliquots have been removed and extracted with 2×5 ml dichloromethane, after different reaction times (t = 1, 6, 12 and 30 min). After the removal of residual substrate and benzoquinone product by extraction, the aqueous phases were treated with excess H 2 SO 4 and TiOSO 4 solutions, and subsequently measured in a quartz cuvette of 1 cm pathlength. Complex concentrations were 5×10 -5 M, the initial H 2 DTBC concentration was 2×10 -3 M. The production of DTBQ has been also determined under identical conditions, but without the extraction step. After a given time interval 1:1 HCl solution was added to stop the reaction, and the solution was mixed with EtOH to obtain 50% EtOH-water mixture.

Superoxide dismutase (SOD) measurements

Superoxide dismutase activity of copper(II) complexes were measured using the McCord-Fridovich reaction (see section 1.4.3.). The formation of the blue product NBTdiformazan ( max = 560 nm) was followed by a Unicam Helios  spectrophotometer, in function of complex concentrations ([Cu 2+ ] tot = 0-1×10 -6 M). In every experiment two reaction mixtures were measured simultaneously along with a blank (without complex) sample, in cuvettes of 1 cm pathlength. The reaction solutions were buffered at pH 7.4 (0.05 M Na 2 HPO 4 /NaH 2 PO 4 ), samples contained 1.010 -4 M NBT and 1.210 -4 M xanthine. Xanthine oxidase enzyme was stored on ice, and was applied in a sufficient quantity for producing A ~ 0.025-0.028 min -1 .

SOD activity was defined using IC 50 values according to relevant literature, which denotes the concentration of the complex at 50% inhibition. %inhibition values were calculated as following:

100 min A min A min A inhibition %                         blank complex blank
When comparing systems studied under different concentration conditions, it must be taken into account that IC 50 is dependent on the used detector (NBT in our case) and its concentration. Therefore, SOD activity may be expressed as k, calculated as [START_REF] Bielski | [END_REF], as later corrected by the authors: http://kinetics.nist.gov/solution/Detail?id=1977BIE/RIC3019-3023:16). However, these k values are only approximate values, since the above k NBT was determined at pH 9.8. Nevertheless, it allows the comparison of SOD activities of different complex systems, regardless of the applied NBT concentrations.

k NBT ×[NBT]/IC 50 (k NBT = 3×10 4 M -1 s -1

Activated phosphoester hydrolytic measurements

Chromophoric phosphoester compounds were used to characterize potential hydrolytic activities of the metal complexes; the activated phosphoesters: BNPPbis(4-nitrophenyl) phosphate, NPP -4-nitrophenyl phosphate, DNPEP -2,4-dinitrophenylethyl phosphate and HPNP -2-hydroxypropyl p-nitrophenyl phosphate substrates were tested in presence of some of the metal-ligand complexes, as hydrolytic catalysts. Aqueous, buffered solutions were used (0.02 M HEPES/CHES, I = 0.1 M (0.08 M NaCl), T = 298 K) for following the reaction by detecting the increase in absorption at 400 nm of 4-nitrophenolate (4NP, ε 400nm = 18900 M -1 cm -1 ) or 2,4-dinitrophenolate in case of DNPEP (ε 400nm = 12100 M -1 cm -1 ). Complex concentrations were set to 1×10 -4 M, initial substrate concentrations were 2×10 -4 M. Data points were collected for 240 min, the autohydrolysis was taken into account at the given pH in every experiments (k obs,corr = k obs -k obs,auto ).

RESULTS AND DISCUSSION

PROTONATION AND COORDINATION PROPERTIES OF TREN3HIS (L 1 )

In the first stages of my PhD work, we were interested in synthesizing free terminal histidine-containing tripodal ligands with C 3 symmetry. The first accomplished preparation resulted the ligand tren3his (L 1 ), bearing solely N-functional groups. Although seven pK values can be expected for tren3his (one tertiary, three primary amino groups and three imidazole rings, Scheme 4.1), only six could be determined in the studied pH range, since the tertiary amino nitrogen probably has a pK < 2, similarly to tren itself [11] (Figure 4.1). Nevertheless, the 1 H NMR spectra of tren3his (Figure 4.2.) show considerable shift between pH 2-6, for both the signals of imidazole rings and the CH 2 protons related to the tren moiety. Consequently, the tertiary amine is involved in a strong H-bonding network with the imidazole rings. Although the imidazole pK values are notably lower than for histamine itself (pK = 6.08, [114]), the values determined are close to those of the related N-()-benzyl substituted derivative [32], and the structurally similar N,N'-di-L-histidylethane-1,2-diamine [114]. Above pH 6 only the CH protons of histidines undergo notable pH-shift (Figure 4.2), hence these processes are related to the deprotonation of primary amino groups. log pK

Protonation of tren3his

H 6 L 1 H 5 L 1 H 2 L 1 H 4 L 1 H 3 L 1
H
C 2 H C 5 H CH CH 2 CH 2 -NHCO CH 2 -N tert dioxane ref. Figure 4.2. pH-dependent 1 H NMR spectra of ligand tren3his ([L 1 ] tot = 0.0019 M, I = 0.1 M NaCl, T = 298 K).

Copper(II) complexes of tren3his

The speciation in the copper(II)-L 1 system proved to be rather complicated. Therefore, beside the combined evaluation of potentiometric and CD data, pH-dependent EPR titrations were also used to uncover the equilibrium properties. The two independent sets of formation constants, derived from our potentiometric/CD and EPR titrations, are in good agreement (Table 4.1). These formation constants are related to a given composition, which in turn may exist in forms of structural isomers. Based on the spectroscopic information discussed later, we were able to identify the main isomers, but due to the polydentate nature of the ligand others with smaller mole fraction may also exist.

CuH 3 L 1 Cu 2+ CuH 2 L 1 CuHL 1 CuL 1 CuH -1 L 1 CuH -2 L 1 Cu 3 H -3 L 1 2 Cu 3 H -4 L 1
a log values derived from the combined potentiometric and CD spectroscopic data b log values derived from the pH-dependent EPR data c EPR parameters of Cu 3 H 2 L 1 2 component spectra; see text in details d during the evaluation of EPR spectra, the formation constants of these species were taken from the potentiometric data, due to their weakly resolved and nearly identical spectra.

In equimolar solution, four differently protonated mononuclear complexes are formed up to pH 8 (Figure 4.3). Both the spectroscopic data ( d-d max = 650 nm, weak positive CD peak at ~ 630 nm, g o = 2.136, A o = 61 G) and the equilibrium constant of the reaction Cu 2+ + H 3 L 1 = CuH 3 L 1 (logK = log 131 -log 031 = 7.92) are consistent with a {3N im } coordination in CuH 3 L 1 [41,65]. In case of peptides with N-terminal histidine, the bis-histamine-like {2NH 2 ,2N im } coordination is preferred in the neutral pH range [42,[START_REF] Sóvágó | Metal complexes of peptides and their derivatives, Biocoordination Chemistry[END_REF]. In principle, this binding mode may appear already in CuH 2 L 1 . However, during the successive deprotonations CuH 3 L 1  CuH 2 L 1  CuHL 1  CuL 1 the visible, CD and EPR spectra all show significant changes (Figures 4.4 Corresponding calculated individual molar spectra can be found in Figure 4.9.

The spectroscopic parameters of CuH 4.1). Since the 6N coordinated tris-histamine-like complex of copper(II) is not known even in the case of histamine [114], the observed spectral changes are due to the replacement of the axially coordinated imidazole ring by the third amino group in CuL At higher pH, further deprotonations were observed leading to the species CuH -1 L 1 and CuH -2 L 1 . Due to the notable stability of CuL 1 (logK = 13.19), these deprotonations take place only above pH 8 (pK CuL 1 = 9.12 and pK CuH -1 L 1 = 10.87), at higher pH than usual for peptides with N-terminal His moieties [38,114]. Such processes can be related to the metal ion promoted deprotonation of amide nitrogen(s), water molecule(s), or, in certain cases, coordinated imidazole ring(s) [START_REF] Gajda | [END_REF]. The increasing negative contribution in the visible CD spectra of CuH -1 L 1 and CuH -2 L 1 indicates metal ion induced deprotonation and coordination of amide nitrogen(s) in our system [117], according to the high affinity of copper(II) toward amide nitrogens. The rather unique visible CD spectrum ('inverse couplet', Figure 4.9 A), as well as the visible and EPR spectra, determined for the species CuH -2 L 1 is nearly identical to those of the CuH -2 L complexes of Ac-HGG-NH 2 , Ac-HGGG-NH 2 , Ac-HGGGW-NH 2 and several other analogs of prion protein octarepeat domain [118][119][120]. Based on this similarity, we suggest {N im ,N -,N tert ,N -} coordination in the equatorial plane of CuH -2 L 1 (Scheme 4.2).

The presence of at least ten potential nitrogen donors in L 1 provides the possibility to the formation of oligonuclear complexes, too. Indeed, at 3:2 metal-to-ligand ratio the solution was clear in the whole studied pH range, but precipitate was observed above pH 6 at 2:1 metal-to-ligand ratio. This observation indicates the formation of trinuclear complexes with Cu 3 H x L 1 2 stoichiometry. The presence of such species, beside the mononuclear complexes, was confirmed by MALDI-TOF MS (Figure 4 ). There are differences, however, between the estimated and measured molecular weights (619.24/620.36 and 1298.41/1301.32), which corresponds to the uptake of one and three protons, respectively. The measured higher molecular weights are due to the reduction of Cu 2+ to Cu + and the corresponding uptake of one proton per metal ion during the ionization process. Identical observations were reported in the literature related to MALDI-TOF MS studies of other copper(II) complexes, formed with cyclen/cyclam derivatives [121], some pseudo-peptides [122] and peptides [123]. Since trinuclear complexes may have several protonation states, the copper(II)-L 1 3:2 system presents a complicated, but rather obvious speciation behavior (Figure 4.3). Cu 3 H 2 L 1 2 is the first trinuclear complex formed around pH 5. Considering the ten available N-donors (the additional two are still protonated), the three copper(II) ions have 3/4N coordination environments, similarly to those in the mononuclear complexes CuH 3 L 1 and CuH 2 L 1 . Indeed, the evaluation of the pH-dependent EPR spectra indicated that the spectrum of Cu 3 H 2 L 1 2 is the superposition of two, rather distinct component spectra having similar g 0 values to CuH 3 L 1 and CuH 2 L 1 (Table 4 1 2 and Cu 3 L 1 2 show comparatively well-resolved hyperfine structures. In Cu 3 L 1 2 the six N-terminal His units create bis-histamine-like coordination environment around the three metal ions. This is similar to that observed in CuL 1 , except the axially coordinated amino group present in the latter. Accordingly, the UV-Vis (and CD) spectra of Cu 3 L 1 2 shows ~ 20 nm blue shift as compared to that of CuL 1 (Table 4.1). Although other alternatives are also possible, the most likely structure of Cu 3 L 1 2 is depicted in Scheme 4.2. In principle, the three legs of L 1 may induce the formation of higher oligomers with the composition (Cu 3 L 1 2 ) n (n > 1), too. However, the linewidth of the isotropic EPR spectra, especially at lower magnetic fields, is sensitive to the radius of the complex molecules. Since intensity loss was not observed, and the linewidth of the EPR spectrum of Cu 3 L 1 2 is similar to that of the analogous Cu 2 (dhen) 2 complex [114], the formation of higher oligomers is at least negligible. Above pH 6, consecutive deprotonations of the trinuclear complexes were observed (Figure 4.3). The characteristic CD spectra of CuH -1 L 1 and CuH -2 L 1 are very similar to those of Cu 3 H -2 L 1 2 and Cu 3 H -4 L 1 2 , respectively (Figure 4.9). Accordingly, in the latter species two copper(II) ions have similar {N im ,N -,N tert ,N -} coordination environment to the corresponding mononuclear complexes. This in turn means that similar deprotonation processes take place at more than one pH unit lower in the trinuclear complexes as compared to the mononuclear ones. This is probably due to the stabilization by the axial nitrogen coordination, which is present only in the mononuclear CuL 1 and CuH -1 L 1 complexes. The fifth deprotonation (Cu 3 H -4 L 1 2  Cu 3 H -5 L 1 2 + H + , pK = 10.76) does not cause considerable change of spectral properties. The central metal ion in the trinuclear complexes, bound to both ligands (Scheme 4.2), has clearly different coordination environment from the other two, since the histamine-like chelate rings belong to two different molecules, and thus fused chelates cannot be formed between them. This coordination mode is identical to that in Cu(His-Gly) 2 complex, which also undergoes a single deprotonation step at high pH [44,[START_REF] Sóvágó | Metal complexes of peptides and their derivatives, Biocoordination Chemistry[END_REF].

 (nm) Cu 3 H 2 L 1 2 Cu 3 L 1 2 Cu 3 H -1 L 1 2 Cu 3 H -2 L 1 2 Cu 3 H -3 L 1 2 Cu 3 H -4 L 1 2 Cu 3 H -5 L 1 2
 (nm) Cu 3 H 2 L 1 2 Cu 3 L 1 2 Cu 3 H -1 L 1 2 Cu 3 H -2 L 1 2

Zinc(II) complexes of tren3his

Potentiometric titrations, MALDI-TOF MS and 1 H NMR measurements were carried out in order to obtain information on the Zn(II)-L 1 complexes. Similarly to the copper(II)-L 1 system, clear solution was obtained in the whole studied pH range up to 3:2 metal-to-ligand ratio, but precipitation was observed at higher metal ion excess. The titration curves of Zn(II)-L 1 0:1, 1:1 and 3:2 systems, along with the formation constants of the metal complexes are presented on Figure 4.10. The evaluation of potentiometric data indicated the formation of complexes with ZnH x L 1 and Zn 3 H x L 1 2 stoichiometries. The presence of such complexes has been verified by MALDI-TOF MS measurements (Figure 4 After three overlapped deprotonations, the major species ZnHL 1 (at pH ~ 5.8) and ZnL 1 (at pH ~ 7.2) are formed. Approaching pH 7, the 1 H NMR spectra show downfield shift upon coordination of both imidazole and CH signals, and an interesting duplication of a tren CH 2 signal at ~ 2.4 ppm can be observed, too (Figure 4.13 dashed frame). This phenomenon is most likely due to the formation of a macrochelate ring associated with a {2N im ,2NH 2 } bis-histamine-like binding, which causes inequivalence of the N tert -CH 2 protons participating in the macrochelate structure. The relations of the deprotonation constants indicate the additional coordination of the third histidine 'pod' in ZnHL 1 and ZnL 1 complexes, too. The pK values of the protonated complexes (pK ZnH2L 1 = 5.42, pK ZnHL 1 = 6.49) are lower than those of the free ligand (pK H2L 1 = 7.21, pK HL 1 = 7.85), and also than the analogous copper(II) containing species (pK CuH2L 1 = 5.55, pK CuHL 1 = 6.92). As a consequence, 5N coordination can be proposed in ZnL 1 (Scheme 4.3). Under basic conditions the complex ZnL 1 releases two additional protons (pK = 8.10 and 10.24). Above pH 7 the mentioned inequivalence of N tert -CH 2 protons ceases, and the signals of CH protons are strongly upfield shifted on the 1 H NMR spectra (Figure 4.13). The formation of slow exchanging complexes was not detected. The coordination of amide nitrogen(s) is indeed unlikely, since N-terminal histidines with {N im ,NH 2 } binding site do not act as anchor for amide deprotonations, and such processes have never been observed in zinc(II)-His-Gly and related systems [42,45,88,124]. These deprotonations are more likely related to the formation of mixed hydroxo complexes (ZnH -1 L 1 = ZnL 1 (OH) and ZnH -2 L 1 = ZnL 1 (OH) 2 ). Interestingly, considerable amount of trinuclear complexes is already formed in equimolar solution, according to potentiometric and NMR data (Figures 4.12-4.14, small peaks around pH 10). This indicates that L 1 is able to bind more than one Zn 2+ with nearly equivalent binding affinities, resulting a significant amount of unbound L 1 above pH 7 in equimolar solution (signals of the free ligand and that of the fast exchanging monocomplexes manifest as average). At 3:2 metal-to-ligand ratio, trinuclear complexes become dominant in neutral and basic solutions (Figure 4.12). At pH 6 the parent Zn 3 L 1 2 is formed; this stoichiometry implies that the zinc(II) ions are coordinated by two arms of the tripod, originated from either the same or two different ligand molecules (Scheme 4.3). Such M 3 L 2 type cluster formation is well described in the literature regarding tripodal ligand complexes [125], and a similar species was detected in the copper(II)-L 1 system, too. Since the metal ions in Zn 3 L 1 2 are coordinated by less nitrogen donors than in ZnL 1 , the trinuclear complex is more prone to deprotonation. The formation of Zn 3 H -2 L 1 2 and Zn 3 H -3 L 1 2 results in considerably broadened 1 H NMR spectra. On the other hand, parallel with the formation of Zn 3 H -4 L 1 2 (pH 8-10) numerous narrow signals appear on the aromatic region (Figure 4.15). The number of peaks cannot be explained by the speciation of the complexes based on the potentiometric model; at pH 10, the observed at least 22 different imidazole signals seem to correspond to a single (de)protonation state (at this pH ~95% of zinc(II) is bound in Zn 3 H -4 L 1 2 ). Among them, at least 7 different C 2 H/C 5 H imidazole proton pairs can be identified on the corresponding TOCSY spectrum (Figure 4.16). The observed multiplication of the signals can imply either the inequivalence of imidazole protons in the trinuclear species due to the conformational differences, or the formation of differently composed (Zn 3n H -4n L 1 2n , n  1) oligomer complexes (Scheme 4.4) with the same metal-to-ligand ratio. The size of such oligomers would be high enough to have detectable variance in their hydrodynamic radii. Accordingly, 1 H DOSY measurements indicate the presence of several complexes with moderately different diffusion parameters, which are, however, clearly distinct from that of the free ligand (at 7.61 and 6.85 ppm, Figure 4.17 Although the presence of several oligomers and the complexity of the NMR spectra do not allow doubtless identification of the coordinating groups and complex compositions, the broadened signals around pH 8 may indicate mixed hydroxo complexes and even their oligomerization through hydroxo bridges. However, it seems clear that the formation of Zn 3 H -4 L 1 2 involves a new type of bridging unit. The TOCSY spectrum (Figure 4.16) indicates that the imidazole C 2 H/C 5 H signals are scattered in a wide range; some of the imidazole signals are considerably upfield-shifted (Figure 4.15). This may imply the presence of imidazolato bridges in these species, participating in the assembly of (Zn 3 H -4 L 1 2 ) n complexes, which results in slow ligand exchange processes.

PROTONATION AND COORDINATION PROPERTIES OF NTA3HIS (L 2 )

The second prepared ligand during my work was the C 3 symmetrical nta3his, the C-terminal counterpart of tren3his. Accordingly, N-and O-donor groups are present in this compound, and the position of the amide group is inversed as compared to L 1 . For the C-terminal histidine containing L 2 ligand (Scheme 4.5), six pK values could be determined between pH 2-11 (Figure 4.18). The deprotonation processes in the acidic and neutral pH range are more separated than that observed in the tren3his system: in the acidic region the carboxylic, above pH 6 the imidazolium groups deprotonate distinctively. The average imidazole pK values of the two ligands (pK im,L 1 = 4.63, pK im,L 2 = 7.11) show notable basicity difference. This indicates that, in the latter case, the imidazolium rings are involved in strong H-bonding network with either the negatively charged carboxylates or the nonbonding electron-pair of N tert . Unlike in case of nta, the protonation of the tertiary amine was not detected in the studied pH range. Interestingly, the nta CH 2 signal also shifts during the deprotonation of the imidazole moiety; the increased electron density on these groups can be explained by a ceasing H-bond between the N tert and imidazolium groups. It is worth mentioning that the amide NH signal is visible almost up to pH 10, indicating unusually slow proton exchange processes for this group.

Protonation of nta3his

Copper(II) complexes of nta3his

In presence of copper(II) ions, L 2 forms mono-and dinuclear complexes at 1:1 and 2:1 metal-to-ligand ratio, respectively. The evaluation of potentiometric measurements did not require the inclusion of trinuclear (3:2) species (Figure 4.20); this itself indicates that nta3his does not provide equivalent binding sites observed for the N-terminal His ligand tren3his. Potentiometric data and pH-dependent UV-Vis spectra were evaluated together in order to calculate the formation constants of the copper(II) species of L 2 : log and pK values are presented in Table 4.2. along with the characteristic UV-Vis and CD spectroscopic parameters of the complexes. In equimolar solution, the visible and CD spectra are practically unchanged between pH 4 and 10 (Figure 4.21), and consequently the calculated individual visible and CD spectra of the species CuHL 2 , CuL 2 , CuH -1 L 2 are identical, and even those of CuH 2 L 2 are very similar (Figure 4.22). This means that the coordination environment of copper(II) in these complexes is also identical, and the four successive deprotonations are related to non-coordinating side chains. Indeed, the pK values of these deprotonations (pK = 3.14, 3.54, 6.20, 7.34, Table 4.2.) are consistent with the proton loss of two carboxylic and two imidazole groups of the free ligand. The 'extra' deprotonation detected for CuH -1 L 2 and its intense CD spectra indicate the presence of a coordinated amide nitrogen in this, and therefore in all mentioned complexes. The spectroscopic parameters of CuH -1 L 2 ( d-d max = 627 nm, g o = 2.1121, A o = 71.5 G, Tables 4.2. and 4.3.) are consistent with a {N tert ,N  ,N im ,O} type coordination with slight rombic distortion, similarly to the CuH 1 L complex of Gly-His [44,127] or glycyl-histamine [128]. The high stability of the Gly-His-like coordination explains the unchanged binding mode during four successive deprotonations of non-coordinated side chains. The formation of the Gly-His-like {N tert ,N  ,N im ,O} coordination mode in the present system takes place at somewhat lower pH (pK CuH 3 L 2 = 3.14) than in the case of Gly-His itself (pK CuL = 4.15 [44]), which can be explained by the important basicity difference of the tertiary (L 2 ) and primary (Gly-His) amines involved in the metal binding. In the copper(II)-Gly-His and related systems, the fourth equatorial position is occupied by a water molecule, which deprotonates with a pK = 9.2-9.4 [START_REF] Sóvágó | Metal complexes of peptides and their derivatives, Biocoordination Chemistry[END_REF]. Such deprotonation was not observed in our system, indicating that the fourth equatorial position in CuH 1 L 2 is more likely occupied by an amide oxygen in chelating position (Scheme 4.6).

Above pH 10, the formation of CuH -2 L 2 results in fundamental changes of the spectroscopic behaviors. On the individual visible spectrum of this species two bands appear with nearly equal intensities at 645 and 765 nm (Figure 4.22. left), suggesting an intermediate structure between square pyramidal and trigonal bipyramidal geometries [129], which is rare in the case of copper(II)-peptide complexes. The intensity of the CD bands increases considerably in the d-d region, indicating the metal coordination of a further amide nitrogen, since only this binding can induce strong chiral perturbation (Figures 4. 21 In general, the amide coordination results in an increase of the EPR parameter A o , due to the increased ligand field around the metal ion. However, in the present case, A o of CuH -2 L 2 (g o = 2.1117, A o = 61.8 G) is considerably smaller than that of the former species, indicating an axially coordinated nitrogen. Considering these facts and the available unbound imidazole rings, a {N tert ,2N  ,N im +N im } type coordination is the most probable in CuH -2 L 2 . The alternative binding mode without the tertiary amine (i.e. {2N  ,2N im }), which is rather common in the case of peptides containing the -HXHsequence, has distinctly different spectral properties [65,91].

The two non-coordinating C-terminal histidine legs in CuH x L 2 (x = 2, 1, 0, -1) obviously may bind a further metal ion. Indeed, our experimental data indicate the formation of four dinuclear complexes above pH 4 (Figure 4.20.) at 2:1 metal-to-ligand ratio. The presence of dicopper(II) complexes was also confirmed by MALDI-TOF MS (Figure 4.24). The obtained isotopic distribution pattern is in good agreement with that calculated for the molecular ion [CuL Considering the high stability of the above-mentioned Gly-His-like {N tert ,N  ,N im ,O} coordination, this binding mode should be preserved in the dinuclear Cu 2 H -1 L 2 species. Indeed, the equilibrium constant for the process CuH -1 L 2 + Cu 2+ = Cu 2 H -1 L 2 (logK = 7.2), as well as the visible and CD difference spectra between Cu 2 H -1 L 2 and CuH -1 L 2 ( d-d max ~ 700 nm, positive CD band around 640 nm, Figure 4.26.) are in accordance with a {2N im ,2COO  } type coordination of the second metal ion in Cu 2 H -1 L 2 [START_REF] Prenesti | [END_REF].

Cu 2 H -1 L 2 -CuH -1 L 2 Cu 2 H -2 L 2 -CuH -1 L 2  (M -1 cm -1
) ) is again in accordance with a {2N im ,N  ,COO  } type coordination [START_REF] Prenesti | [END_REF] in the equatorial plane of the second copper(II) (Scheme 4.6). This species has surprisingly high stability: it is the dominant complex over nearly 3 pH units. The next deprotonation (pK Cu 2 H -2 L 2 = 9.35) induces further characteristic changes on CD spectra both in the UV and visible region (Figure 4.26), which reflects the metal coordination of the third amide nitrogen. On the other hand, the visible spectrum shows ~ 20 nm red shift between pH 8-10.3 (Figure 4.25). The spectral changes exclude the formation of a binding mode present in CuH 2 L 2 . The third amide nitrogen binds most likely to the second metal ion, creating a {N tert ,N  ,N im ,O; 2N  ,2N im } coordination mode in Cu 2 H -3 L 2 . Peptides with -HXH-sequence undergo two strongly overlapping amide deprotonations, due to the preferred formation of a {2N  ,2N im } coordinated species [65,91]. However, in the present case the two successive amide deprotonations are well separated, because they are involved in a considerably less favored, eight-membered chelate ring. Moreover, this chelate ring contains the metal-bound tertiary amine, too, which probably makes the conformational change needed for the Cu 2 H -2 L 2  Cu 2 H -3 L 2 + H + deprotonation more difficult, resulting in the dominant formation of Cu 2 H -2 L 2 at pH 8.

(nm) Cu 2 L 2 Cu 2 H -1 L 2 Cu 2 H -2 L 2 Cu 2 H -

Zinc(II) complexes of nta3his

Potentiometric and pH-dependent 1 H NMR measurements were performed to study the forming L 2 complexes in presence of Zn(II). Surprisingly, precipitate formation was observed already at 1:1 metal-to-ligand ratio, between pH 6 and 9, even at ~1 mM complex concentration. Clear solutions could be obtained at 3.5-fold ligand excess, however, bis-or tris-ligand complexes were not needed to describe the titration curves. This, and the fact that the observed pH of the precipitation is too low for the hydrolysis of Zn(II), all suggest that the precipitate corresponds to weakly soluble zinc(II)-ligand complex(es), which can only be studied in diluted solutions.

Table 4.4. presents the formation constants of zinc(II)-L 2 complexes, calculated on the basis of titrations at 1:3.5 metal-to-ligand ratio; Figure 4.27. shows the distribution of the species at such ligand excess (up), and at a simulated 1:1 metal-to-ligand ratio (down). By the latter simulation, it is clear that the observed precipitation in equimolar solution is most likely related to the ZnL 2 complex (involved pH region presented as gray field). Since a great ligand excess would have resulted nearly identical NMR spectra to that of the free ligand, pH-dependent 1 H NMR measurements were carried out at 1:1 zinc(II)-toligand ratio; the samples were centrifuged in the precipitated pH regions, and spectra of the clear solutions were recorded. Considerable intensity loss was observed for all the signals on the pH-dependent spectra (Figure 4.28); nevertheless, we could obtain structural information related to the zinc(II) complexes of L 2 . Based on potentiometric measurements, complex formation starts above pH 5. Around this pH, the broadening of the NMR signals can be observed; moreover, around neutral pH the CH and CH 2 signals undergo considerable changes (CH of the free ligand was not observed at all, see Figure 4.19). All these observations indicate that in the forming complexes, Zn(II) coordination involves mostly the His moiety of the ligand (N im and possibly COO -groups). At higher pH, the broadened imidazole peaks slightly sharpen and multiply, indicating the deceleration of the ligand exchange in the forming species. This phenomenon becomes dominant above pH 9, following the deprotonation ZnL -13.24 (7) Zinc(II)-dependent NMR spectra recorded around neutral and basic pH support these conclusions. At pH 7.9 a single, widened C 5 H signal can be observed (Figure 4.29. down) indicating intermediate ligand exchange rate, implying complex(es) with multiimidazole coordination. Approaching pH 10, this structure transforms to result a slow exchanging species (Figure 4.29. up), most likely related to the Gly-His-type {N im ,N -,N tert } binding, since deprotonation of coordinated water molecules does not cause such deceleration of the ligand exchange process. H 2 N-XH-type sequences are known to promote this Gly-His-type coordination: the deprotonation of the amide NH was observed in Zn(II)-Gly/Ala-His systems [45,46,126]. Therefore, the formation of such species is not surprising; however, it becomes dominant at much higher pH as compared to the analogous copper(II) complex (pK ZnL 2 = 8.74, pK CuH 2 L 2 = 3.53). This indicates that the multiimidazolic environment competes strongly with the Gly-His binding mode up until basic pH. However, it remains unexplained, how or why this multiimidazole coordination triggers precipitation in the neutral pH range. Based on the tendency of symmetric tripodal ligands towards oligomerization [125], the most likely answer is the polymerization-aggregation of the complex, resulting in significant decrease of its solubility.

PROTONATION AND COMPLEX FORMATION OF NTA1HIS-NH 2

In the second part of my PhD work, our attention turned to unsymmetrical, partially functionalized tripodal ligands. Synthetic experiments were attempted in solution and solid phase as well. The latter case resulted the mono-His functionalized C-terminally free (nta1His) and protected (nta1His-NH 2 ) ligands (Scheme 4.7). Unfortunately, these compounds turned out to be unstable for long-term investigations (see Experimental section 3.1.4), therefore experimental data are limited mainly to potentiometric characterization of the ligands, as well as copper(II) and zinc(II) complex formation with the ligand nta1His-NH 2 .

Protonation of the ligands

The ligands nta1his and nta1his-NH 2 have three detectable pK values in the studied pH range (Table 4.5). The most basic deprotonation, observed around pH 7 in both cases, most probably belongs to the proton loss of the imidazolium group. Interestingly, another relatively high pK was observed in both ligands (pK H2L = 6.44 and 5.66), which is probably not related to carboxylic groups. More likely they correspond to the proton loss of the N tert -H + group, which points to an important difference between these systems and the trishistidyl-functionalized nta derivative (nta3his); the protonation of N tert was not observed between pH 2-10.5 in the latter case (see Section 4.2.1). This elevated pK can be explained by the presence of the free carboxylate groups of the nta moiety, being able to participate in a N tert ---H + ---COO -type hydrogen bonding system. Undoubtedly the same effect causes the increased acidity of the nta -COOH groups; the most acidic carboxylic groups are already deprotonated below pH 2 in both ligands.

Copper(II) and zinc(II) complexes of the ligand nta1his-NH 2

Unfortunately, the instability of the ligand did not result accurate experimental data regarding the nta1his-metal systems, therefore complex formation could not be investigated. However, freshly prepared solutions of nta1his-NH 2 were appropriate for solution equilibrium studies, allowing to gain some information on complexation properties of mono-His functionalized nta derivatives. Both mononuclear copper(II)-and zinc(II) complexes were detected in ESI-MS for this ligand, too (see Appendix Figures A. The distribution of the copper(II)-and zinc(II)-nta1his-NH 2 species is depicted in Figure 4.30, the calculated stability constants and deprotonation constants can be found in Table 4.6. The coordination behavior of the ligand is very similar towards Cu 2+ and Zn 2+ ions: the same complexes are formed with different relative stability. In presence of copper(II) ions, CuHL and CuL species are formed at acidic pH, but their formation overlaps with that of the dominant CuH -1 L species. Spectrophotometric titration of the complex solution allowed the calculation of individual molar UV-Vis spectra for the main complexes. The absorption of CuHL manifests at relatively low energies, suggesting N im and COO -coordination; the  with that of the CuH -1 L 2 complex of nta3his (615 nm, Figure 4.22). The structural analogy between the two systems and the high stability of the 'extra' deprotonated complex suggests the same, Gly-His-type {N im ,N -,N tert ,O} structure. This species deprotonates only above pH 10, most probably related to proton loss of a coordinated water molecule; the process does not cause significant perturbation in the coordination sphere (Figure 4.31). In presence of Zn 2+ ions, the protonated ZnHL complex forms in dominant quantities between pH 3-5; its deprotonation leads to the species ZnL and ZnH -1 L, almost in a co-operatively fashion. Without any experimental proof, we can only assume that the highly stable Gly-His-type structure is preserved in the ZnH -1 L complex as well. Its formation takes place more than two pH units higher than in case of the analogous copper(II) complex, which is in agreement with the higher preference of copper(II) towards amide N -coordination.

PROTONATION AND COORDINATION PROPERTIES OF TREN2HIS (L 3 ) AND TREN1HIS (L 4 )

The most recently synthesized tripodal ligands are the asymmetric, N-terminally free His-containing tren2his (L 3 ) and tren1his (L 4 , Scheme 4.8). These two compounds, along with tren3his, serve as a decent example for studying the impacts of gradual functionalization of tripodal platforms on metal complex formation. The protonation constants and pK values of tren2his and tren1his ligands derived from pH potentiometric titrations are collected in Table 4.7. The identification of the deprotonating groups has been supported by pH-dependent 1 H NMR measurements (Figures 4. 32 The pK of the tertiary amino group could be determined only for L 3 , which was also supported by the pH-dependent 1 H NMR spectra (Figure 4.32): considerable shifts can be observed for the signals related to the tren moiety in the acidic pH range. Such displacements of 1 H NMR signals was not observed in case of tren1his (Figure 4.33), which suggests low basicity for the tertiary nitrogen in L 4 , similarly to tren itself [11].

Protonation of tren2his and tren1his

Copper(II) complexes of tren2his

The ligand tren2his forms both mono-and oligonuclear copper(II) complexes, depending on the metal-to-ligand ratios. The distribution curves of the detected species are depicted on Figure 4.34, their formation constants and the spectroscopic parameters of the main species are listed in Table 4.8.

In equimolar solution only mononuclear complexes are formed. CuH 2 L 3 is the first major species around pH 4, its protonation state suggests 3N coordination. Indeed, the equilibrium constants for the process Cu 2+ + H 2 L 3 = CuH 2 L 3 (logK = 10.26), its UV-Vis spectrum ( d-d max ~ 640 nm), as well as the relatively weak positive CD band between 600-800 nm are in perfect agreement with {2N im ,NH 2 } coordinated complexes of several -HXH- [124] or -HXXH- [88] peptides. Approaching neutral pH, the next deprotonation results in the formation of CuHL 3 . In this species only the tren-like NH 2 group is protonated, i.e. the metal ion has the highly stable bis-histamine-type {2N im ,2NH 2 } coordination (Scheme 4.9). This binding mode is supported by spectral similarities with the analogous bis-histamine coordinated complexes: the detected d-d transitions ( d-d max = 640 nm) and CD spectra (intense positive CD band at 660 nm and a negative band around 300 nm, Figure 4.36. B) are nearly identical to those observed in the Cu(II)-His-Gly [130], Cu(II)-His-Xaa-Xaa [118], Cu(II)-N,N′-dihistidylethane-1,2-diamine [114] and in the strongly related Cu(II)-tren3his systems.

The tren-like amino group in the bis-histamine coordinated structure of CuHL 3 is too far from the metal ion to be axially coordinated. Nevertheless, the pK values of the subsequent two deprotonations steps are more than 2 units lower than the corresponding pK (= 9.59) in the free ligand. These facts and the fundamental spectroscopic changes during the consecutive deprotonation steps (CuHL A) indicates considerable increase of the ligand field around the metal ion, i.e. 4N coordination with at least one amide nitrogen. Indeed, since the free ligand is non-protonated around pH 10, the protonation state of the complex suggests one coordinated amide nitrogen. Considering the preferred formation of fused chelate rings, too, a {NH 2 ,N  ,N tert ,NH 2 tren } coordination mode can be assumed in CuH 1 L 3 (Scheme 4.9). Another evidence of the suggested coordination mode arises from the similarities of CD spectra observed for CuH -1 L 3 and that reported for the CuH 2 L complex of the HGG peptide [118], where the chiral perturbation arises from the {NH 2 ,N  } chelate ring. It must be pointed out that the proposed {NH 2 ,N  ,N tert ,NH 2 tren } coordination mode in CuH -1 L 3 leaves a histidyl-functionalized arm free, with an unbound histamine-type binding site. As it was previously observed in case of the ligand tren3his, a free N-terminal His arm induces dimerization processes at metal ion excess via the formation of Cu 3 H -x L 2 type complexes, due to the high stability offered by the bis-histamine-type binding. Accordingly, the presence of trinuclear complexes was detected by MALDI-TOF MS (Figure 4.35) at 3:2 copper(II)-to-ligand ratio. Indeed, the evaluation of our potentiometric data indicated the formation of two trinuclear species (Cu 3 H 1 L 3 2 and Cu 3 H 2 L 3 2 ), as their consideration was found to be necessary for the successful fitting of the titration curves performed at Cu(II)-L 3 3:2 ratio. However, these species do not form in significant quantities at 1:1 or 2:1 metal-to-ligand ratios, because of the high stability of both monoand dinuclear complexes of L 3 ; these were also detected by MALDI-TOF MS (Figure 4.35).

3  CuL 3  CuH -1 L 3 ,
The obtained isotopic distributions are in agreement with that calculated for the onepositively charged complexes [Cu II H 1 L 3 ] + , [Cu II 3 H 5 L 3 2 ] + and [Cu II 2 H 3 L 3 ] + . The reduction of the copper(II) centers and the concomitant proton uptakes take place in this copper(II)-ligand system, too, resulting [Cu I L 3 ] + , [Cu I 3 H 2 L 3 2 ] + and [Cu I 2 H 1 L 3 ] + species, as it was also observed during MALDI-TOF MS analyses of Cu(II)-L 1 and -L 2 complexes (Sections 4.1.2. and 4.2.2). The fact that in equimolar solutions the dinuclear complexes form only in small quantities indicates that L 3 provides two coordination sites for copper(II) with rather different metal binding affinities. At 2:1 metal-to-ligand ratio, the deprotonation of the first appearing minor dinuclear species Cu 2 L 3 results in the formation of Cu 2 H -1 L 3 (Figure 4.34. down), which predominates in the solution between pH 5-8. Taking into account that in the same pH range the bis-histamine-like {2N im ,2NH 2 } coordination provides high stability in equimolar solution, the conservation of this binding site in Cu 2 H 1 L 3 is safe to assume. In this way, the four N donor groups of the tren-like subunit are preorganized to bind a further metal ion, and the {NH 2 tren ,N tert } chelate may act as an anchor to promote the deprotonation and coordination of the amide nitrogen(s). The protonation state of Cu 2 H 1 L 3 implies that the second copper(II) is bound to either a {N  ,N tert ,NH 2 tren } or {N  ,N tert ,N  } donor set (with protonated tren-like amino group in the latter case, see Scheme 4.9). Assuming that the {2N im ,2NH 2 } coordinated metal ion in Cu 2 H 1 L 3 has similar component Vis spectrum than that of CuHL 3 , the Cu 2 H 1 L 3 CuHL 3 difference spectrum may be informative regarding the coordination environment around the second metal ion. This subtraction resulted a 'usual' Vis spectrum (Figure 4.36. A) with a relatively high energy d-d maximum ( d-d max = 546 nm), which favors the {N  ,N tert ,N  } coordination in the equatorial plane of the second copper(II). Although, none of the above mentioned two donor sets can be ruled out, the latter one may also explain the practically unchanged UV-Vis and CD spectra during the next deprotonation, i.e. deprotonation of the tren-like NH 3 + group without metal ion assistance. In fact, the processes Cu 2 H 1 L 3  Cu 2 H 2 L 3  Cu 2 H 3 L 3 are overlapped (Figure 4.34. down), therefore a further deprotonation takes place nearly parallel. For this latter process, proton loss of either a coordinated water molecule or imidazole ring can be taken into account. In principle, both may induce structural rearrangement which would explain the observed changes on the UV-Vis and CD spectra of Cu 2 H 3 L 3 (Figure 4.37).

 (nm) Cu2L Cu2H-1L Cu2H-2L Cu2H-3L A Cu 2 L 3 Cu 2 H -1 L 3 Cu 2 H -2 L 3

Zinc(II) complexes of tren2his

Zinc(II) complexes of L 3 were characterized by pH potentiometric, 1 H and 2D NMR measurements. Water soluble complexes were found to be forming even at 2:1 zinc(II)-toligand ratio, indicating the presence of both mono-and dinuclear complexes. The formation constants of the complexes are listed in Table 4.9, their speciation is depicted in Figure 4.38. In the equimolar system, mononuclear zinc(II) complexes dominate in the whole studied pH range. The complexation starts with the formation of ZnH 2 L 3 at pH 4.5. The 1 H NMR spectra around pH 5 show notable broadening for the imidazole proton signals (Figure 4.39), indicating their role in the metal binding. Since both imidazole rings and one of the -amino groups are deprotonated by metal ion assistance in ZnH 2 L 3 , histamine-type binding with the additional coordination of the second imidazole ring {2N im ,NH 2 } can be assumed in this species. The deprotonation of ZnH 2 L 3 (pK = 6.07) results in the formation of ZnHL 3 , a dominant complex between pH 6-8. The Zn(II)-assisted deprotonation of the remaining CH-NH 3 + results in the formation of bis-histamine-type {2N im ,2NH 2 } coordination, which involves a macrochelate ring formation between the two His units (Scheme 4.10). The formation of this macrochelate is favorable, since the basicity corrected stability constant of ZnHL 3 (logK = log ZnHL 3 -log H 5 L 3 = -17.13) is more than 4 units higher than that of the analogous Zn(histamine) 2 complex (logK = log ZnL 2 -2log H 2 L = -21.71 [114]). The presence of this macrochelate, similarly to the zinc(II)-tren3his system (see The 'extra' deprotonation observed for the oligonuclear species between pH 7-8 could be assigned to (i) deprotonation of coordinated water molecule(s), (ii) metal-promoted deprotonation of amide nitrogen(s) or (iii) formation of -imidazolato-bridge between two zinc(II) ions. Among these processes only (ii) and (iii) may lead to slow ligand exchange processes and notable shift of imidazole proton signals. It is worth to mention that the strongly related N,N'-di-L-histidylethane-1,2-diamine (dhen) ligand also forms imidazolato-bridged oligozinc(II) complexes in the same pH range [114]. The increased electron density on the imidazolate ring obviously results in remarkable upfield shift of C 2 H signals (up to ~2.2 ppm [114,131,132]), as compared to the free ligand. On the other hand, in the Zn(II)-GlyHis system [126] the coordination of imidazole ring and the deprotonation of nearby amide nitrogen also resulted in 0. The bis-histamine-type coordination of zinc(II) in ZnHL 3 provides a preorganized tren-like {NH 2 ,N tert ,2N -} binding site for the second metal ion (Scheme 4.10), i.e. the deprotonation of the amide nitrogens is promoted by the anchoring {NH 2 tren ,N tert } chelate.

This binding mode explains the strongly cooperative deprotonations during the process ZnHL 3 + Zn 2+ = Zn 2 H -2 L 3 + 3H + (Figure 4.38. down), and also the fact that dinuclear complexes are formed only at metal ion excess, in parallel with the deprotonation of NH 2 of the free tren arm. However, the two imidazole moieties in Zn 2 H -2 L 3 cannot exist in three chemically different environments with nearly equal populations; therefore, more than one species must be present under these conditions. In order to explain the above-mentioned observations, we assume a dimerization process of 2Zn

Copper(II) complexes of tren1his

The mono-histidyl functionalized tren1his forms only mononuclear complexes in presence of copper(II). The determined formation constants and spectroscopic parameters are listed in Table 4.10, the distribution curves of the copper(II)-L 4 species are depicted in Figure 4.44. The equilibrium constant for the reaction Cu 2+ + H 2 L 4 = CuH 2 L 4 (logK = 8.24), as well as the individual UV-Vis ( d-d max ~ 670 nm) and CD ( max ~ 720 nm) spectra (Figure 4.45.) of CuH 2 L 4 complex suggest histamine-like {N im ,NH 2 } coordination in this species (Scheme 4.11), similarly to several -HXX-peptides [118,124].

The formation of CuHL 4 , which is the dominant species around pH 5, induces remarkable changes in the spectroscopic properties indicating a characteristically different binding mode. In principle, several binding modes are possible in this species, but the intense CD spectrum (Figure 4.45.) excludes the possibility of the sole coordination of the achiral tren-subunit {NH 2 tren ,N tert ,NH 2 tren }. In fact, the observed 'inverse couplet' on the CD spectrum of CuHL 4 is unique, and characteristic for the species in which the chiral perturbation arises from a single histidine unit bound to copper(II) in a seven-membered {N im ,N  } chelate. This type of coordination results in a very similar CD spectrum for the copper(II) complexes of Ac-HGG-NH 2 , Ac-HGGG-NH 2 , Ac-HGGGW-NH 2 and several other analogs of prion protein octarepeat domain [118][119][120], and also for the {N im ,N - ,N tert ,N -} coordinated CuH -2 L 1 species in the copper(II)-tren3his 1:1 system (Section 4.1.2, Figure 4.9. A). Accordingly, in CuHL 4 we suggest {N im ,N -,N tert ,NH 2 tren } coordination (Scheme 4.11). The participation of a tren-like NH 2 and tertiary amino groups in the coordination is dictated by the protonation state of the complex and the enhanced stability of fused chelate rings, respectively. Indeed, the 4N-coordination is also supported by the relatively high energy of d-d transitions ( d-d max ~ 585 nm) and the corresponding EPR parameters (see Appendix, Table A Above pH 5, two overlapping deprotonations lead to CuH -1 L 4 , which is the only species present in solution above pH 8. The UV-Vis, CD and EPR spectra indicate considerable rearrangement of the coordination sphere during these deprotonations: the d-d transitions are red-shifted by 47 nm, the g values are nearly unchanged, but A z is considerably decreased (178  157 G, Appendix Table A.1). The 'inverse couplet' on the CD spectra also disappears during the CuHL 4  CuL 4  CuH -1 L 4 transformation. These facts indicate the coordination of a further N-donor in axial position, and therefore changes in the coordination geometry. Tren derivatives are known to prefer pentacoordinated structures, and the spectroscopic data are in accordance with the formation of square pyramidal geometry. In one of the possible structures the equatorial binding sites of CuHL 4 are retained, and the second tren-like NH 2 group is coordinated in the apical position (Scheme 4.11), i.e. the NH 3 + deprotonates without metal ion assistance.

Zinc(II) complexes of tren1his

In the Zn(II)-L 4 system, four differently protonated mononuclear species could be identified by potentiometry (Table 4.11, Figure 4.47). At the beginning of complex formation (pH 4.5-5.5) all 1 H NMR signals related to the histidine unit are significantly broadened (Figure 4.48). Even though ZnH 2 L 4 complex is not produced in higher quantities than 40%, the selective broadening on the 1 H NMR spectra indicates that zinc(II) is bound to the histidine moiety in a histamine-like binding mode ({N im ,NH 2 }, Scheme 4.12). The logK value for the process Zn 2+ + H 2 L 4 = ZnH 2 L 4 (logK = log ZnH 2 L 4 -log H 2 L 4 = 4.51) also supports this type of coordination [114]. The formation of ZnH -1 L 4 , a dominant complex between pH 7-10, occurs through three strongly overlapped deprotonations (ZnH 2 L 4  ZnH -1 L 4 + 3H + ), which are so cooperative that ZnL 4 could not be detected at all. In parallel (pH 6.0-7.5), remarkable changes can be observed on the NMR spectra. All CH 2 signals first broaden then sharpen to result a complex spectrum in the basic pH range (Figure 4.48. A,D). The source of this complicated peak set is the slow ligand exchange rate of ZnH -1 L 4 , and the inequivalence of all CH 2 protons related to the metal-bound ligand. The slow ligand exchange observed for ZnH -1 L 4 indicates that the 'extra' deprotonation is related to the formation of amide-bound species (and not to mixed hydroxo complexes) [126]. Beside the amide group, the complete formation of ZnH -1 L 4 at pH 7 suggests zinc(II) protomoted deprotonation and coordination of all three primary amino groups (their pK values in the free ligand are 7.07, 9.04, 9.90). Although the tertiary amino group is deprotonated at pH 7 even in the absence of zinc(II), the preferred formation of fused chelate rings and the inequivalence of all tren-CH 2 protons support its coordination. The simultaneous coordination of amide-, NH 2 -and imidazole nitrogens is unlikely in the case of N-terminal His fragments [42,133], therefore the axial coordination site can be occupied by either the N im or the NH 2 group. Considering the thermodynamically favorable 5-membered {N -,NH 2 } chelate ring and the above-mentioned deprotonation processes, we suggest {3NH 2 ,N -,N tert } type binding in ZnH -1 L 4 (Scheme 4.12). In consequence of the saturated coordination sphere in ZnH 1 L 4 , the pK of the subsequent deprotonation is rather high (10.73), since the coordinating hydroxide ion should displace one of the bound nitrogens.

ASSESSMENT OF THE METAL BINDING PROPERTIES OF TRIPODAL PEPTIDES

An important aim of this work was to enhance the metal binding capabilities of linear peptides with the assistance of functionalized tripodal platforms. The comparison of metal ion sequestering ability of different ligands based on simply the formation/stability constants, however, can be misleading, due to the different basicity of the compared ligands. In order to demonstrate the increased metal ion sequestering ability of our tripodal ligands, predominance diagrams were simulated in presence of our ligand systems, and also of other structurally relevant ligands.

The impact of gradual functionalization of tren scaffold on metal ion affinity can be well studied in the trenXhis (X = 1, 2, 3) series. Based on the predominance diagrams in Cu(II)-and Zn(II)-trenXhis equimolar systems (Figure 4.49), it can be stated that the metal ion is bound most importantly by tren3his (L 1 ) in the acidic-neutral, and by tren1his (L 4 ) in the basic pH range. This tendency stands for both copper(II) and zinc(II) complexes. Interestingly, tren2his (L 3 ) does not become dominant metal binding ligand next to the other two at any pH. These findings point to the two highly dominant binding modes in the trenXhis series, which in fact predominate in different pH ranges. The bis-histamine-type binding -supplemented by axial coordination in case of L 1 -is major binding site up to neutral pH, while the involvement of the tren-type binding site (N tert , NH 2 and/or N - groups) becomes more and more important in the basic pH range with decreasing functionalization of the tripodal scaffold. The ligand tren1his can be considered as a 'maximum' in copper(II) binding capability in this ligand series above pH 7: simulations demonstrated that its copper complexes dominate even over copper(II)-tren species at 1:1:1 Cu(II)-L 4 -tren ratio. Even though the two symmetrically functionalized tren3his and nta3his (L 2 ) ligands are very different in terms of basicity and nature of donor groups, their metal binding capabilities are worth comparing (Figure 4.50). L 2 bears carboxylic groups which are capable of binding copper(II) ions strongly at relatively low pH. In agreement with the previous statements, the bis-histamine-type coordination provides exceptional binding site for copper(II) in the acidic-neutral pH range. As this binding mode loses significance above pH 8, the nta3his ligand becomes the dominant copper(II) binding agent. Based on these observations, we can state that the Gly-His-type {N im ,N tert ,N -,O} structure of nta3his is not able to compete with the bis-histamine-type coordination, but provides a more stable coordination environment than that of the {N im ,N -,N tert ,N -} structure in CuH -2 L 1 . [

Cu(II)] tot = [L 1 ] tot = [L 2 ] tot = 0.001 M.
Considering the structural organization of the metal binding moieties provided by the tripodal scaffold, it might not be surprising that the two symmetrical tris-His ligands demonstrate higher metal binding abilities in the whole pH range than that of their 'structural models', histamine and Gly-His (Figure 4.51). This dominance is so important that the Zn(II) complex of histamine does not produce in notable quantities in the Zn(II)-L 1 -histamine 1:1:1 equimolar system. If we mean to draw conclusions on enhanced metal binding with respect to linear peptides, probably the most convincing example is the comparison of our systems with the simplest H 2 N-XXH-type peptide ligand, Gly-Gly-His (GGH), which can be considered as one of the strongest copper(II) binding peptide sequence providing {NH 2 ,N -,N -,N im } binding site in aqueous solutions [134]. Simulated predominance curves of Cu(II)-L 1 -L 3 -L 4 1:1:1:1 system show that the copper(II) complex of GGH does not form in dominant quantities at any pH (Figure 4.52). In fact, the presence of 1 eq. GGH does not alter significantly the distribution of copper(II) among the trenXhis ligands, implying that it is unable to compete in copper(II) binding with the above-described, highly stable coordination environments provided by the trenXhis series. This observation proves convincingly that metal binding capabilities of peptides can be significantly improved by the functionalization with tripodal scaffold.

CATECHOL OXIDASE MIMICKING WITH THE METAL-LIGAND

COMPLEXES

One of our main goals was to explore the effect of increasing histidyl functionalization of tripodal platforms on enzyme mimicking properties of the formed copper(II) complexes. To this end we tested our copper(II) complexes as catechol oxidase mimics. For that purpose, we chose the widely investigated 3,5-di-tert-butylcatechol (H 2 DTBC) substrate, which is selectively converted to 3,5-di-tert-butyl-o-benzoquinone (DTBQ) in presence of copper(II) containing catalysts and dioxygen. Catechol oxidases are type-3 copper enzymes [48,135], therefore the copper(II) complexes of trenXhis (X = 1, 2, 3) ligands are well suited for such biomimetic studies, and provide a decent possibility for structure/nuclearity/activity comparison.

We performed detailed kinetic measurements in order to establish kinetic parameters (Section 4.6.1), whereas substrate binding studies helped us to reveal mechanistic information (Sections 4.6.2-4.6.4). Experimental conditions of kinetic studies are collected in Table 4.12; further experimental details can be found in Section 3.3.1.

Kinetic study of Cu(II)-L 1 , -L 2 and -L 3 systems

In case of all copper(II)-ligand systems, pH-dependent kinetic measurements were carried out to reveal the pH optimum of the reaction; further experiments were performed at this pH, in buffered solutions. Of all investigated systems, the copper(II) complexes of tren3his (L 1 ), nta3his (L 2 ) and tren2his (L 3 ) ligands were found to possess high catechol oxidase activity. In every cases, the multinuclear (Cu(II)-L 1 3:2, Cu(II)-L 2 and -L 3 2:1) complexes showed the highest catechol oxidase mimicking capability. Although we observed activity for the Cu(II)-L 1 1:1 system in the basic pH range, the presence of trinuclear complexes in solution makes it impossible to distinguish the catalytic performance of the mono-and the trinuclear species. Therefore, further kinetic experiments were performed only at 3:2 copper(II)-to-ligand ratio, where trinuclear complexes dominate in the studied pH region. 4.12.

The pH corr -k obs,corr profiles of catalytic H 2 DTBC oxidation are presented on Figure 4.53. In case of Cu(II)-L 1 3:2 system we observed a reproducible local maximum around pH 7.8, and increasing rate constants above pH 8.3. In presence of Cu(II)-L 2 2:1 system, a continuous increase of rate constants was observed above pH 7, and their values are higher at any pH than in the former case. Both Cu(II)-L 3 1:1 and 2:1 systems resulted a pH optimum (pH corr 7.4 and 8.7, Figure 4.53), however the observed reaction rates are very different in the two cases. The speciation of the complexes in 50% ethanol-water solvent mixture is generally similar to those determined in pure aqueous solutions, thus the observed catalytic activities are mainly related to the complexes Cu 3 H -1 L 1 2 /Cu 3 H -3 L 1 2 , Cu 2 H -3 L 2 , CuL 3 and Cu 2 H -2 L 3 . Based on these data (and to avoid the high rate of autooxidation of H 2 DTBC), the further kinetic measurements have been performed at pH corr = 7.8 (L 1 ), 8.9 (L 2 ), 7.4 (L 3 mononuclear system) and 8.7 (L 3 dinuclear system). 4.12. Solid lines correspond to fitted curves. The initial reaction rates of the catalytic oxidation as a function of H 2 DTBC concentration show saturation kinetics above 10-50-fold excess of substrate over the complexes (Figure 4.54). This indicates a fast pre-equilibrium between the complex and the substrate molecules before the rate determining redox processes, therefore the Michaelis-Menten enzyme kinetic model is applicable in our systems. Table 4.13. lists the calculated Michaelis-Menten parameters, along with those of some previously reported model complexes. Although the comparison with literature data is complicated by the many different conditions used, it can be stated that the multinuclear complexes of L 1 , L 2 and L 3 possess high activities, outperforming the few copper(II)-peptide complexes studied earlier [65,80]. It is interesting to note, that in all cases amide coordinated complexes are the pre-active species for H 2 DTBC oxidation (further discussion in Section 4.6.4).

Regarding the non-peptide complexes, the k cat values determined for our copper(II)ligand systems can be considered average; the activities are ~4 orders of magnitude lower than that of the native enzyme isolated from sweet potato [47]. However, the k cat /K M ratios, especially in case of Cu(II)-L 2 2:1 and Cu(II)-L 3 2:1, support the high catalytic efficiency of the present systems: the k cat /K M determined for Cu(II)-L 3 2:1 system exceeds the same value of the catechol oxidase model possessing the highest k cat value reported so far (see Table 4.13). This elevated efficiency can be attributed to the impressively strong substrate binding ability of the tripodal peptide complexes (i.e. low K M ), representing a unique and very advantageous feature among catechol oxidase mimicking model complexes. We also studied the performance of our complexes in longer time intervals, in presence of ~120 eq. excess of substrate ([complex] tot = 1×10 -5 M). After 1 hour, experimental turnover numbers of 41.1 h -1 (Cu(II)-L 1 3:2, pH 7.8) and 64.1 h -1 (Cu(II)-L 2 2:1, pH 8.9) was found, however these data are strongly limited by the spontaneous diffusion of air into the studied solution.

a ligand B1: N',N'-bis(2-pyridylmethyl)-N,N-(2-hydroxybenzyl)(2-hydroxy-3,5-di-tert-butylbenzyl)-1,3-propanediamin-2- ol; ligand B2: N-(2-hydroxybenzyl)-N',N',N-tris(2-pyridylmethyl)-1,3-propanediamin-2- 20 [71] ibCO j (6.5) 2293 2.5×10 -3 917200 H 2 O, 20 [47]
In order to have more insights into the mechanism of H 2 DTBC oxidation, reaction rates were measured as a function of complex and dioxygen concentration under pseudo-first order conditions. In all cases first order dependence was observed (Figures 4.55 and 4.56), which is in agreement with many other catecholase model systems [56,61,66,67]. According to the Michaelis-Menten model, the observed pseudo-second order rate constant (k', slope of the complex concentration dependent reaction rate curves) is equal to k cat /(K M + [S]). The values calculated from the Michaelis-Menten parameters (k' calc,L 1 = 13.9 M -1 s -1 , k' calc,L 2 = 670.5 M -1 s -1 , k' calc,L 3 = 154.4 M -1 s -1 ) are close to the measured ones (k' L 1 = 7.97 M -1 s -1 , k' L 2 = 495.9 M -1 s -1 and k' L 3 = 272 M -1 s -1 ), which confirms the validity of our applied kinetic model. 4.12.

Substrate binding study in Cu(II)-L 1 3:2, Cu(II)-L 2 2:1 and Cu(II)-L 3 2:1 systems

After studying the kinetic aspects of catalytic H 2 DTBC oxidation by our complexes, we wished to know more about the factors influencing the catalytic cycle, and attempted to draw the mechanism of oxidation. Therefore, substrate binding and mechanistic studies have been carried out concerning the catalytically active Cu(II)-L 1 3:2, Cu(II)-L 2 2:1 and Cu(II)-L 3 2:1 systems.

The three metal centers in Cu 3 H x L 1 2 complexes may raise the question about the number of bound catechols in the fast pre-equilibrium step. Therefore, we studied the binding of 4-nitrocatechol (H 2 4NC), a widely used chromophoric probe to mimic catecholmetal ion interaction [139,140]. Since this electron-poor catechol ligand possesses higher redox potential than H 2 DTBC, its metal ion binding can be easily monitored by UV-Vis spectroscopy under aerobic conditions. First, the pK values and the spectral properties of H 2 4NC have been determined in 50% EtOH/H 2 O solvent (Table 4.14). The pK values of this ligand are considerably lower than those of H 2 DTBC, thus the formation of ternary species with Cu 3 H x L 1 2 complexes is expected at lower pH. pH-dependent absorption spectra of Cu(II)-L 1 -H 2 4NC ternary systems were recorded at different complex-to-H 2 4NC ratios; Figure 4.57. shows the change of absorbance at 434 nm in function of pH. At 1:1 complex-to-H 2 4NC ratio, the pK of H 2 4NC deprotonation is considerably shifted to the acidic region (from pK = 6.78 to ~4.5), clearly indicating direct binding of 4-nitrocatechol to the complexes resulting in metal-induced deprotonation. With decreasing concentrations of the trinuclear complexes, the deprotonation of H 2 4NC follows a two-step process indicating that only one equivalent catecholate is coordinated under our conditions; the excess H 2 4NC deprotonates at higher pH, without the participation of the copper(II) complexes. Since the coordination of the analogous monodentate 4-nitrophenol was not observed in a similar experiment, bidentate coordination of catechol substrates can be suggested.

We have also investigated the binding of H 2 DTBC applying inert (argon) atmosphere in order to prevent the catalytic oxidation of the substrate. Figure 4.58. shows the UV-Vis spectra of Cu(II)-L 1 -H 2 DTBC ternary system at pH 7.8 with increasing H 2 DTBC concentrations. Three new bands appear on the electronic absorption spectra at 280, 300 and 440 nm. The wide band at 440 nm and the shoulder around 300 nm are related to the ternary species Cu 3 H x L 1 2 (DTBC 2-) in agreement with earlier reports on the spectral behavior of Cu 2+ -ligand-catecholate ternary complexes [61,71], while the band at 280 nm belongs to the absorption of H 2 DTBC itself. The d-d absorption band of Cu(II) around 625 nm is almost unchanged during the experiment, only a slight blue shift indicates the alteration of the coordination sphere around the metal ion. In addition, the characteristic absorption band of the oxidized 3,5-di-tert-butyl benzoquinone (DTBQ) product does not appear around 400 nm. These facts indicate that under anaerobic conditions the formation of the copper(II)-catecholate ternary species is not followed by the reduction of Cu 2+ ions and the oxidation of the substrate. Similar findings were observed during the analogous experiment in presence of the Cu(II)-L 3 2:1 system. Substrate dependent UV-Vis spectra (Figure 4.59. A) show two overlapping bands appearing between 350 and 450 nm upon addition of increasing amount of H 2 DTBC, while the d-d band of Cu(II) shifts towards the higher energies (from ~580 to 564 nm, Figure 4.59. A). Similarly to the Cu(II)-L 1 3:2 system, neither the strong absorbance of DTBQ at 400 nm (which would have resulted an absorbance of ~1 within the applied experimental conditions), nor the reduction of copper(II) were found. Instead, the absorption bands (between 350-450 nm) attributed to the CT-band of copper(II)-bound catecholate [61,72,141,142], and the absorbance of the free (unbound) monodeprotonated HDTBC  , present in small quantities (pK H 2 DTBC = 10.35 in 50/50% MeOH/H 2 O medium [143]), can be observed on the UV-Vis spectra.

The CD spectra, reflecting exclusively the environment of the metal ions, also show profound changes upon addition of H 2 DTBC (Figure 4.59. B), indicating important rearrangement of the coordination sphere upon substrate binding. Since the chiral perturbation arises exclusively from the ligand L 3 , the CD spectra directly prove the formation of Cu(II)-L 3 -substrate adduct. Based on the CT-bands related to the Cu(II)-ligand-catecholate ternary adducts (UV-Vis absorbance at 440 nm and CD band at 448 nm in Figures 4.58. and 4.59. B inserts, respectively), we could estimate the apparent stability constant (K app ) of the complexsubstrate adduct according to the equilibrium processes Cu 3 H w L 1 2 + H 2 DTBC = Cu 3 H x L 1 2 (DTBC 2-) and Cu 2 H y L 3 + H 2 DTBC = Cu 2 H z L 3 (DTBC 2-). The non-linear regression of the data resulted K app = 3570 M -1 and 14200 M -1 for the Cu(II)-L 1 3:2 and Cu(II)-L 3 2:1 systems, respectively (see solid lines of Figures 4.58. and 4.59. B inserts). These values are in good agreement with the results of our saturation kinetic measurement (1/K M = 8928 M 1 and 27624 M 1 in case of Cu(II)-L 1 3:2 and Cu(II)-L 3 2:1 systems, respectively), since the Michaelis constant corresponds to the dissociation constant of the above-mentioned adduct. It is worth mentioning that the extraordinary high binding ability of Cu(II)-L 3 2:1 system is not restricted to H 2 DTBC; similarly high K app values (> 20000 M -1 ) were determined for catechol and 4-nitrocatechol, too, by similar CD measurements (see Appendix A.24).

Based on these results, the role of dioxygen is the oxidation of the substrate activated by the oligonuclear copper(II)-L 1 and -L 3 complexes, and not the regeneration of the Cu 2+ species (re-oxidation of Cu + ), as it was proposed for the native enzyme [48,49], and for many catalytic dinuclear copper(II) complexes [52]. In fact, the observed phenomenon is in agreement with an oxidation pathway pursued by mononuclear copper(II) complexes [61,76], involving the formation of radical intermediate(s) (see later). Anaerobic UV-Vis measurements on the H 2 DTBC binding were carried out in the Cu(II)-L 2 2:1 system, too. Figure 4.60. shows the recorded spectra in presence of 0 to 3 eq. H 2 DTBC. The observations are fundamentally different from the previous systems: the strong absorbance of DTBQ at 400 nm appeared immediately after the introduction of 1 eq. substrate in absence of dioxygen and the intensity of the Cu(II) d-d band decreased. Though this decrease of Cu(II) band intensity is undeniable, its entire disappearance does not occur, i.e. the redox reaction is not stoichiometric. This is consistent with many previous observations that the reduction of copper(II) under anaerobic conditions is not complete until the introduction of considerable excess of H 2 DTBC [66,79]. These results indicate a single-step two-electron oxidation of the catechol substrate by the two cooperating copper(II) centers in the Cu(II)-L 2 complexes, which is the main proposed mechanism for binuclear catecholase-like copper(II) complexes [52].

Peroxide, o-benzoquinone and radical formation in Cu(II)-L 1 and -L 2 systems. Mechanism of catecholase-like activities in the studied Cu(II)-L systems

The detection and quantification of H 2 O 2 has been accomplished by treating the aqueous phase with acidic TiO 2+ , after the elimination of the organic compounds by dichloromethane extraction. The concentration of peroxotitanyl complex can be measured spectrophotometrically at 408 nm. The formation of DTBQ and H 2 O 2 has been determined parallel during the complex catalyzed reaction of H 2 DTBC. Both Cu(II)-L 1 and -L 2 catalytic systems show initial accumulation of H 2 O 2 in the beginning of the reaction, followed by its slow decay (Figure 4.61). Moreover, the concentration of the formed DTBQ exceeds the peroxide levels considerably in both cases. This suggests a dynamic equilibrium of H 2 O 2 between its formation and consumption after the initial stages of the catalytic reaction. As described above, the formation of copper(II)-catecholate unit was observed in the Cu(II)-L 1 -H 2 DTBC system (Figure 4.58.) under anaerobic conditions, without the subsequent redox reaction between Cu(II) and the substrate. This is consistent with mononuclear catalytic centers, where valence tautomer (VT) equilibrium between the copper(II)-catecholate and copper(I)-semiquinone adduct is strongly dependent on the donor groups and structure of the applied ligand [52,144]. Both tautomer forms are oxygen sensitive, therefore the catalytic oxidation should proceed through the formation of radical intermediates [54] (semiquinone (SQ), superoxide radical or both), which might be detected by EPR. Although copper(II)-superoxide complexes are generally EPR silent due to the antiferromagnetic interaction [145,146], the formation of SQ intermediates has been previously observed by EPR [56,64,70,76]. For that reason, we attempted to detect the formation of radical intermediate(s) in both aerobic and anaerobic conditions. In the absence of dioxygen radicals were not detected, according to the anaerobic UV-Vis measurement of the Cu(II)-L 1 -H 2 DTBC system. Under aerobic conditions, however, a well-defined doublet EPR signal (g = 2.0046±0.0001, a H = 2.67 G, Figure 4.62.) was observed, similarly to those detected earlier for intermediates of H 2 DTBC oxidation [64,70,76]. Hence, we must conclude that in the copper(II)-L 1 -H 2 DTBC system the copper(II)-bound catecholate is oxidized only in the presence of dioxygen, through the formation of oxygenated radical intermediate (Scheme 4.13).

Based on the detailed kinetic study and careful evaluation of the substrate binding experiments, the oxidation of H 2 DTBC catalyzed by the oligonuclear copper(II) complexes of L 1 and L 2 proceeds via one-and two-electron transfer from the catechol to the copper(II) ion(s), respectively. Scheme 4.13. presents the suggested basic reaction steps of the catalytic oxidation of H 2 DTBC. Under the conditions used for L 1 (pH 7.8, Cu(II)-L 1 3:2) trinuclear complexes (mostly Cu 3 H -1 L 1 2 ) are the pre-active species. Nevertheless, the oxidation of H 2 DTBC proceeds in a one-electron transfer pathway, with the participation of a single Cu 2+ center. The other metal ions might have only inferior contribution in substrate binding, which is probably due to the spatial Cu(II)-Cu(II) separation in these trinuclear species (Scheme 4.2). In this system the copper(II)catecholate/copper(I)-semiquinone valence tautomer (VT) equilibrium is shifted towards the copper(II)-catecholate form. One of these tautomers reacts with dioxygen in the rate determining step, resulting the formation of oxygenated radical intermediate(s) [54]. After a subsequent intramolecular electron transfer DTBQ and H 2 O 2 are released, regenerating the copper(II) center and allowing the coordination of a new substrate molecule.

In contrast, the oxidation in the Cu(II)-L 2 -H 2 DTBC system is catalyzed by two co-operating copper(II) centers, similarly to the catechol oxidase enzymes. Even though dicopper(II) species are formed already at pH 4 in Cu(II)-L 2 2:1 system, the tendency of catechol oxidase-like activity follows the formation of Cu 2 H -3 L 2 . After coordination of the substrate to the dicopper(II) core, DTBQ and dicopper(I) species are formed in a fast electron transfer process. The reduced metal centers are re-oxidized by dioxygen, yielding a Cu(II)-O 2 2--Cu(II) species, as it is often proposed in the literature [52]. This in turn reacts with an incoming second substrate molecule, and the dicopper(I) species is regenerated after releasing the second DTBQ and H 2 O 2 (via proton transfer).

Since hydrogen peroxide does not seem to be produced in stoichiometric quantity in either catalytic system, the participation of H 2 O 2 in the catalytic reaction can be suggested. Most probably, as a widely known oxidizing agent, the peroxide molecule might contribute to the reoxidation of Cu(I) center(s) to Cu(II), as it was previously described for other catalytic copper(II) complexes [79,147]. It is somewhat surprising that the observations in Cu(II)-L 3 2:1 system correspond more to the mechanistic behavior of Cu(II)-L 1 3:2, rather than that of the other dinuclear Cu(II)-L 2 system. Instead of the two-electron transfer between the coordinated substrate molecule and the copper(II) centers, only the formation of the complex-substrate intermediate was observed under inert conditions. Thus, the involvement of the Cu(II)-catecholate/Cu(I)semiquinone valence tautomerism (VT) is safe to assume in the present case, too. The presence of dinuclear complexes corresponds to the formation of a mixed-valence intermediate, which would suggest that H x DTBC binds to only one copper ion. A similar Cu(II)Cu(II)-catecholate/Cu(II)Cu(I)-semiquinone VT was already reported in the literature [56]. Indeed, the absorption band observed between 350-450 nm (Figure 4.59. A) is in agreement with bidentate catecholate binding, however the observed very low K M may indicate that the second copper(II) somehow assists in the substrate binding [148]. As it is suggested in the Cu(II)-L 1 system, the VT-equilibrium is strongly shifted toward the copper(II)-catecholate state, since neither appreciable decrease of d-d band intensity, nor the EPR spectrum of semiquinone radical was observed under anaerobic conditions. Nevertheless, in presence of dioxygen the ternary adduct reacts with dioxygen in a fast rate determining step, resulting the formation of oxygenated radical intermediate(s) [54] and a subsequent intramolecular electron transfer, while DTBQ is formed and the copper(II) state is regenerated.

Catechol oxidation: linear vs. tripodal peptide complexes

In the past years several biochemically relevant peptide models were investigated in our research group, focusing on their coordination chemistry [43,65,88,90,[START_REF] Gajda | [END_REF]149]. These sequences were proved to play important roles in metal binding in native biomolecules, contributing to the function of the relevant metalloproteins in e.g. metal transport [149] or enzymatic processes [43,88,90]. By studying the redox mimicking capabilities of such strong metal binding sequences, we might gain information and estimations on redox activities of other, biologically relevant peptide fragments. Thus, it is important and desirable to reveal the redox activities of such peptide-copper(II) species. Copper(II) complexes of several linear peptides (P1-P5, Scheme 4.14.) have been investigated as catechol oxidase mimics. These sequences, containing at least three histidyl residues, serve as decent ground of comparison with our histidine-containing tripodal peptide complexes.

In equimolar copper(II)-peptide solutions, all complexes were found to catalyze the oxidation of H 2 DTBC above pH 7. Unlike the rate of autooxidation, which increases exponentially, the complex catalyzed reaction rates show maximum curves in function of pH, since the differently protonated species exhibit different catalytic efficiency (Figure 4.63). The observed pH maxima are between pH 8.2-9.8. The pH corr -k obs,corr profiles of the studied peptide complexes, along with the reaction rate curves in function of H 2 DTBC concentration are depicted in Figures 4. 63. and 4.64, respectively. The non-linear regression of the data resulted the kinetic parameters listed in Table 4.15. As it can be seen, the pre-active copper(II) species are in all cases imidazole and amide nitrogen coordinated complexes, mostly in a {2N im ,2N -} environment. This is somewhat surprising, since the increasing ligand field strength induced by the coordination of amide nitrogens, in principle, stabilizes the +2 oxidation state of copper and consequently weakens the redox activity of the complexes. Still, similar findings have been observed in earlier reports [81,150], and we also found that the catalytically active tripodal peptide-copper(II) complexes are amide N -coordinated species (see Section 4.6.1). This seemingly general behavior prompted us to assume that the deprotonated amide nitrogens might contribute to the catalytic performance of the complexes by acid-base catalysis, i.e. by abstracting protons from the catechol -OH groups, and thus facilitating substrate binding (Scheme 4.15). Since the complexes of multihistidine peptides studied here or earlier are considerably more active than those of the N-terminal -synuclein fragments containing no histidine [80], the +1 oxidation state of copper is likely stabilized by the coordination of 2-3 imidazole rings, similarly to that suggested for the interaction of Cu(I) with A peptides [151].

With this knowledge, it is worth comparing the kinetic behavior of linear and tripodal peptide complexes, with respect to the product formation rate (k cat ), stability of the complex-substrate adduct (K M ) and pH optimum. In cases where the catalysis involves one copper(II) center coordinated by tripodal peptides (Cu(II)-L 1 3:2 and Cu(II)-L 3 1:1), the pH optima of the reaction are considerably lower (pH < 8) than that of the linear peptide systems. While k cat values of linear peptides are relatively high (k cat of Cu(II)-P3 1:1 approaches that of the dinuclear Cu(II)-L 3 system), their K M value remains in the 10 -3 -10 -4 M range, causing generally lower k cat /K M ratio as compared with Cu(II)-tripodal peptide systems. Thus, we can conclude that the structure organizing effect of tripodal peptides influences advantageously the substrate binding and therefore, overall catalytic efficiency in catechol oxidation.

SUPEROXIDE DISMUTASE (SOD) MIMICKING WITH THE METAL-LIGAND COMPLEXES

The investigated tripodal and linear peptide-copper(II) complexes demonstrated strong ability for dioxygen activation. Such oxidative reactions may involve the formation of superoxide radical, too. Copper(II) complexes of His-containing linear peptides are wellknown superoxide dismutase mimicking compounds [82,88,90,91,150,152,153]. Therefore, we also studied the superoxide dismutating ability of copper(II) complexes formed with the tripodal and linear His-peptides, thus gaining a deeper insight into their redox enzyme mimicking properties.

All measurements were carried out around neutral pH (7.0-7.8), in phosphate buffered solutions (I = 0.1 NaCl, T = 298 K). Superoxide radicals were generated by the xanthinexanthine oxidase system, inhibition and IC 50 values were determined by following the reduction of nitrotetrazolium Blue choride (NBT) at 560 nm, in presence and absence of the copper(II) complexes.

The experiments require extremely low complex concentrations (~10 -7 M order of magnitude), therefore simulations have been performed in order to discover the distribution of the complex species under such conditions. The calculations revealed that trinuclear complexes of L 1 do not form in dominant quantities, therefore SOD activity was determined for the monocopper(II) complexes of tren3his only. At two-fold metal ion excess, dinuclear complexes of nta3his and tren2his were found to be dominant even at such low complex concentration levels, allowing the establishment of their superoxide dismutase activity. The presence of free copper(II) ion was negligible in all Cu(II)-ligand systems.

All investigated systems, except for Cu(II)-L 4 , showed considerable SOD-like activity. Copper(II) complexes of L 4 did not present any catecholase-like activity either, thus we can assume that the saturated, highly stable coordination sphere around the metal ion in CuH -1 L 4 (Scheme 4.11.) prevents the coordination of the substrate in these catalytic tests. This finding is supported by the fact that the inhibition levels observed during our SOD experiments are even lower than that determined for free copper(II) in aqueous solutions [153].

The inhibition curves resulted by the tripodal peptide-copper(II) systems are depicted in Figure 4.65. The obtained kinetic parameters (IC 50 and k) are presented in Table 4. 16, together with that of the studied linear peptide-copper(II) systems, the native Cu,Zn-SOD and free copper(II) as comparison. Based on the determined k values (which are independent of the applied NBT concentration) the highest SOD-mimicking capabilities are presented by the dinuclear complexes of L 3 . Mononuclear copper(II) species of this ligand were also found to be able to dismutate superoxide radicals with high efficiency, however, the dicopper(II) complexes do not present two-fold increase in k over the mononuclear complexes, indicating different activities regarding the two copper cores, which is in agreement with the two copper centers being in very different coordination environments. The observed activity is on the same level as the most active studied linear peptide system, the -H(Xaa) n HXaaH-type sequence P5, having a {2N im ,2N -} coordinated copper(II) in the relevant pH range [149]. The other linear peptide-copper(II) species have lower SOD activities, falling behind that of the other two tripodal ligand-copper(II) systems.

Altogether, it can be stated that the observed SOD activities for the copper(II)-peptide systems are significant, especially that of the tripodal peptide complexes. However, all of the obtained IC 50 or k values are more or less similar; they are in the same order of magnitude, outperforming the activity of the free copper(II) phosphate solutions itself, but not approaching the efficiency of the native Cu,Zn-SOD enzyme isolated from bovine erythrocytes [65] (see Table 1.2. in Section 1. 4.3). This indicates that the tripodal structure of the ligands does not affect considerably the catalytic performance of the copper(II) species in superoxide dismutation function, unlike as it was observed in the oxidation of H 2 DTBC, where the tripodal platforms were found to enhance notably the substrate binding capabilities of the peptide complexes.

SUMMARY

In my PhD thesis, I present synthesis and characterization of tripodal peptide-type ligands, along with the extensive study on their complex formation properties in presence of copper(II) and zinc(II) ions, natural cofactors of metalloenzymes. By using His-functionalized tripodal platforms, our aim was to enhance the metal binding capabilities of linear peptide ligands, with respect to stability and structure. We applied the metal complexes of the ligands in catalytic model reactions such as catechol oxidation, superoxide dismutation or phosphoester hydrolysis, in order to establish their enzymemimicking behavior. Beside kinetic characterization, detailed substrate binding and mechanistic studies have been carried out, especially focusing on catechol oxidation by the copper(II)-ligand complexes.

The studied ligands provided the opportunity to (i) compare the coordination chemical properties of tripods bearing C-and N-terminal histidine units, as well as to (ii) study the effect of gradually increasing number of N-terminal His moieties. Thanks to the high number of available donor groups, formation of multinuclear species could be induced by the ligands, and some interesting self-assembling features were observed as well.

The scientific content and value of this work can be summarized in the following points:

(1) The firstly synthesized, free N-terminal histidine containing ligand, tren3his (L 1 ), forms mono-and trinuclear (M 3 L 2 type) complexes in presence of both metal ions. The coordination properties are directed by the highly stable bis-histamine-type {2N im ,2NH 2 } coordination. Complexes with this binding mode dominate in equimolar metal-ligand solutions up to pH 8. In the basic pH range, metal induced deprotonation of the amide nitrogens takes place in presence of copper(II), while zinc(II) complexes deprotonate to form mixed hydroxo species. At 3:2 metal-to-ligand ratio, dimerization of the mononuclear units occurs with the assistance of a third bound metal ion, resulting M 3 H -x L 1 2 type species. NMR study of the trinuclear zinc(II) complexes formed at pH 10 revealed that these complexes may be involved in even higher level of assembly, forming Zn 3n H -x L 1 2n oligomer structureswhile EPR measurements did not suggest such self-assembly in presence of copper(II) ions.

(2) The C-terminal histidine containing nta3his ligand (L 2 ) presents different coordination properties towards copper(II) and zinc(II) ions. In the copper(II)-L 2 system mono-and dinuclear complexes could be detected. The spectroscopic data are consistent with a highly stable, Cu(II)-Gly-His-like, {N tert ,N -,N im ,O} type coordination, dominating in the equimolar solution until pH 10. Above that, this structure transforms into the fivecoordinate CuH -2 L 2 complex, having an intermediate geometry between trigonal bipyramid and square pyramid. The Gly-His-type binding mode allows the binding of a second copper(II) ion at metal excess; the imidazole and carboxylic groups participate in this 106 coordination in the acidic-neutral, while amide nitrogens in the basic pH range. In presence of zinc(II) ions, the water insoluble ZnL 2 complex forms between pH 6 and 9, causing precipitation in a wide pH range. Zn 2+ -dependent 1 H NMR spectra indicated different ligand exchange rates in the neutral and the basic pH range; the deceleration of the ligand exchange processes at basic pH implies the formation of the Gly-His-type {N im ,N -,NH 2 } coordination in ZnH -1 L 2 . Accordingly, the precipitating species should be coordinated by imidazole/COO -groups, which leads, probably due to oligo-/polymerization, to significant decrease in solubility.

(3) In the second part of the work, asymmetrically functionalized tripodal peptides have been synthesized and studied. The bis-histidyl tren derivative tren2his (L 3 ) forms monoand dinuclear complexes in presence of copper(II) and zinc(II) ions. At 1:1 metal-to-ligand ratios, the bis-histamine-type coordination is predominant until neutral pH. In basic solutions, amide nitrogen and tertiary nitrogen coordinated copper(II) species, as well as mixed hydroxo zinc(II) complexes formed, similarly to the tren3his ligand systems. In presence of 2 eq. copper(II), Cu 2 H -x L 3 (x = 1, 2, 3) species dominate above pH 4. The formation of dinuclear species is related to the presence of two basically different metal binding sites in the molecule: aside of the bis-histamine-type coordination site, the primary tren NH 2 , the tertiary amino group and two amide nitrogens act as secondary binding site at the center of the ligand. In presence of 2 eq. copper(II), the complex Cu 2 H -1 L 3 dominates over 4 pH units. The dizinc(II) complexes have slow ligand exchange rate on the NMR time scale; the spectra indicated interesting dimerization processes, where the imidazole ring might act as bridging ligand.

(4) The mono-His-functionalized tren derivative tren1his (L 4 ) forms only mononuclear complexes with both metal ions. In presence of copper(II) ions, CuHL 4 and CuH -1 L 4 complexes are the major species with {N im ,N -,N tert ,NH 2 } and {N im ,N -,N tert ,2NH 2 } coordination, respectively. The latter complex is five-coordinated, similarly to tren-metal complexes, but the peptide-type functionalization causes considerable distortion in geometry. In presence of Zn 2+ ions, the analogous ZnH -1 L 4 complex predominates in the whole pH range; the related 1 H NMR spectra is surprisingly complicated, due to the slow ligand exchange and the magnetically inequivalent CH 2 protons. The coordination sphere of the MH -1 L 4 complexes is completed by an axially coordinating histidine-nitrogen donor group.

(5) Catechol oxidase mimicking properties of the investigated copper(II)-ligand systems have been studied from kinetic and mechanistic aspects. We observed significant catalytic activities in the oxidation of 3,5-di-tert-butylcatechol (H 2 DTBC) in case of copper(II)-L 1 3:2, copper(II)-L 2 2:1 and copper(II)-L 3 2:1 systems. Reaction rates were measured in function of pH, substrate, complex and oxygen concentrations. Substrate-dependent measurements resulted saturation-like behavior in all ligand systems, allowing the Michaelis-Menten enzyme kinetic treatment. In all cases, first order dependence was detected in the reaction rates regarding oxygen and complex concentrations. Substrate binding and other mechanistic studies revealed that Cu(II)-L 2 2:1 system carries out twoelectron oxidation of H 2 DTBC, similarly to the native enzymatic pathway. The Cu(II)-L 1 3:2 and Cu(II)-L 3 2:1 systems, despite the tri-/dinuclear pre-active complex, perform oneelectron oxidation of the substrate via the formation of a semiquinone type radical intermediate. (6) Connecting structure and activity, comparison has been made between catechol oxidase mimicking complexes of tripodal and linear peptides. As a general feature, coordinated amide nitrogens were suggested to facilitate substrate binding by the abstraction of protons from the -OH groups of the incoming substrate, serving as supporting acid-base catalytic sites in catecholase mimicking Cu(II)-peptide complexes. Furthermore, we have found that the structure organizing effect of the tripodal scaffold positively influences the substrate binding capabilities of the complexes, and therefore, overall catalytic efficiency. The studied tripodal ligands favor the formation of multinuclear species, further improving catalytic properties by allowing two-electon oxidation pathways: our dinuclear systems demonstrated impressively high k cat /K M values in H 2 DTBC oxidation, even among non-peptide-type catecholase model complexes. (7) The superoxide dismutase (SOD) mimicking properties of linear and tripodal peptide-copper(II) complexes were also studied. Except for copper(II)-L 4 , all investigated copper(II)-ligand systems showed significant superoxide dismutating ability in the modified McCord-Fridovich indirect test reaction. Their IC 50 values are in the same range as those determined in histidine containing linear peptide-copper(II) systems; however, their activity is still almost two order of magnitudes lower than that of the native Cu,Zn-SOD.

In summary, we synthesized and investigated a new group of tripodal peptide ligands bearing histidine moieties. With thorough and systematical solution equilibrium and spectroscopic studies, we uncovered the driving forces of metal coordination, and established the impact of the tripodal structure in stability, structure and catalytic properties of the forming complexes. Our results confirm the beneficial effect of tripodal scaffolds in peptide-type ligand-metal complexes, and emphasize the possibilities lying within tripodal peptides in the field of enzyme mimicking.

ÖSSZEFOGLALÁS

Doktori értekezésemben peptid típusú tripodális ligandumok előállítását és jellemzését, illetve a metalloenzimekben nagy számban előforduló réz(II)-és cink(II)ionokkal alkotott fémkomplexeik tanulmányozását mutatom be. A tripodális állványzat alkalmazásával a lineáris peptidek fémkötő képességét szerettük volna továbbfejleszteni, szerkezeti és stabilitási szempontból. A képződött fémkomplexeket katalitikus modellreakciókban alkalmaztuk (pirokatechin oxidáció, szuperoxid dizmutáció, illetve foszfátészter hidrolízis), és jellemeztük enzimutánzó sajátságukat. A kinetikai vizsgálatok mellett részletes szubsztrátkötési vizsgálatokra, illetve mechanisztikus tárgyalásra is sor került, főként a réz(II)komplexek pirokatechin oxidáz aktivitására vonatkozóan.

A vizsgált ligandumok jó összehasonlítási alapként szolgáltak (i) az N-és C-terminális hisztidint tartalmazó tripodális vegyületek összevetéséhez, illetve (ii) a fokozatosan növekvő számú hisztidin-alegységet tartalmazó tripodális peptidek tanulmányozásához koordinációs kémiai szempontból. A nagyszámú donorcsoportok jelenlétének köszönhetően többmagvú fémkomplexek képződése is előidézhető volt, illetve érdekes asszociációs tulajdonságok is megfigyelhetők voltak.

A munka tudományos tartalma összefoglalható az alábbi pontokban:

(1) Az elsőként előállított és vizsgált, N-terminális hisztidint tartalmazó tren3his ligandum (L 1 ) mindkét fémion jelenlétében egy-és hárommagvú (M 3 L 2 típusú) komplexeket képez. A koordinációs sajátságokat a nagy stabilitású bisz-hisztamin {2N im ,2NH 2 } típusú kötésmód határozza meg; az ilyen szerkezetű fémkomplexek ekvimoláris oldatukban pH 8-ig dominánsak. A lúgos pH tartományban réz(II)ionok jelenlétében amidnitrogén koordináció, míg a cink(II)-ligandum rendszerben vegyes hidroxo komplexek képződése zajlik. 3:2 fém-ligandum aránynál a monomer egységek dimerizációja következik be egy harmadik fémion megkötésével, M 3 H -x L 1 2 összetételű komplexek képződéséhez vezetve. A cink(II)-ligandum 3:2 rendszerben pH 10-en felvett NMR spektrumok arra utaltak, hogy ez a folyamat még ennél is nagyobb mértékű, Zn 3n H -x L 1 2n típusú oligomerizációhoz vezethet. Az ESR eredmények nem utaltak hasonló mértékű asszociációra a megfelelő réz(II)komplexek esetében.

(2) A C-terminális hisztidint tartalmazó nta3his (L 2 ) ligandum eltérő koordinációs viselkedést mutat réz(II) és cink(II)ionok jelenlétében. A réz(II)-ligandum rendszerekben egy-és kétmagvú komplexek képződése volt észlelhető. A spektroszkópiás vizsgálatok egy nagy stabilitású, a réz(II)-Gly-His rendszerre jellemző {N tert ,N -,N im ,O} koordinációs környezetre utalnak, mely ekvimoláris oldatban pH 10-ig uralkodó kötésmód. Ez egy érdekes, ötös geometriával rendelkező CuH -2 L 2 komplexszé alakul át, trigonális bipiramis és négyzetes piramis közötti szerkezettel. A Gly-His típusú koordináció lehetővé teszi egy második fémion megkötését a ligandum által, mely savas pH tartományban imidazol és karboxilát, míg lúgos tartományban amidnitrogének által koordinált. Cink(II)ionok jelenlétében csapadékképző, alacsony oldhatóságú ligandum komplexek képződnek pH 6 és 9 között. Cink(II)-függő 1 H NMR spektrumok a ligandumcsere sebesség megváltozására utaltak a semleges és lúgos oldatokban; a lassú cseresebesség itt is a Gly-His-szerű {N im ,N -,NH 2 } koordinációt valószínűsíti. Ezek alapján a semleges tartományban jellemző csapadékképző komplexben imidazol-és karboxilátcsoportok kötődnek, melyek, talán oligo-/polimerizáció révén, az oldhatóság jelentős csökkenéséhez vezetnek.

(3) A munka második felében aszimmetrikusan funkcionalizált tripodális peptideket állítottunk elő és vizsgáltunk. A bisz-hisztidil tren származék tren2his (L 3 ) egy-és kétmagvú komplexeket képez mind réz(II), mind cink(II)ionok jelenlétében. 1:1 fémligandum arányoknál a bisz-hisztamin típusú koordináció semleges pH-ig domináns. Bázikus oldatban amidnitrogének és a tercier nitrogén által koordinált réz(II)komplexek, illetve vegyes hidroxo cink(II)komplexek képződnek, hasonlóan a tren3his rendszerekhez. A kétmagvú komplexek kialakulása két, jól elkülöníthető fémkötő hely jelenlétéhez rendelhető a molekulában: a bisz-hisztamin-szerű kötőhely mellett a tren primer és tercier aminocsoportja, és a két amidnitrogén szolgáltat egy második fémkötő helyet. Az így képződő rézkomplexek közül a Cu 2 H -1 L 3 részecske 4 pH egységen keresztül domináns. A kétmagvú cink(II)komplexek lassú ligandumcseréjűek az NMR időskálához mérve; a spektrumok érdekes dimerizációs folyamatra utaltak, ahol az imidazolgyűrű hídligandumként betöltött szerepe is felmerül.

(4) A mono-His funkcionalizált tren származék tren1his (L 4 ) csak egymagvú komplexeket képez mindkét fémionnal. Réz(II)ionok jelenlétében a CuHL 4 és a CuH -1 L 4 részecskék dominálnak, az utóbbi a tren-hez hasonló, ám az amidnitrogén koordináció miatt jelentősen torzult ötös geometriával rendelkezik. A cink(II)-ligandum rendszerben a megfelelő ZnH -1 L 4 komplex az egész pH tartományban domináns; a részecske NMR spektruma meglepően bonyolult, amit a lassú ligandumcsere sebesség és a különböző CH 2 csoportok protonjainak mágneses inekvivalenciája okoz. Az MH -1 L 4 komplexek koordinációs szféráját egy hisztidin nitrogén axiális koordinációja egészíti ki.

(5) Jellemeztük a tanulmányozott réz(II)-ligandum rendszerek pirokatechin oxidáz utánzó sajátságait kinetikai és mechanisztikus szempontból. Jelentős katalitikus aktivitás volt tapasztalható a réz(II)-L 1 3:2, réz(II)-L 2 2:1 és réz(II)-L 3 2:1 rendszerekben, a 3,5-di-terc-butil pirokatechin (H 2 DTBC) szubsztrát oxidációjában. Vizsgáltuk a reakciósebességeket a pH, illetve a szubsztrát-, komplex-és oxigénkoncentráció függvényében. A szubsztrátfüggő mérések telítési kinetikai viselkedést mutattak minden esetben, így alkalmazható volt a Michaelis-Menten kezelésmód. Minden esetben elsőrendű függést állapítottunk meg a reakciósebesség és a komplex-/oxigénkoncentráció között. Mechanisztikus és szubsztrátkötési kísérletek rámutattak, hogy a Cu(II)-L 2 2:1 rendszer kételektronos oxidációs mechanizmus szerint működik, hasonlóan a natív enzim mechanizmusához. Ezzel szemben a Cu(II)-L 1 3:2 és Cu(II)-L 3 2:1 rendszerek, a többmagvú preaktív fémkomplexek ellenére egyelektronos oxidációs mechanizmust követnek, szemikinon típusú átmeneti komplex képződésén keresztül.

(6) Hogy kapcsolatot teremtsünk szerkezet és aktivitás között, összehasonlítottuk tripodális rendszereink pirokatechin oxidáz aktivitását néhány lineáris peptid komplexének aktivitásával. Általánosan megállapítható, hogy a pirokatechin oxidáz utánzó réz(II)-peptid komplexekben a koordinált amidnitrogén kiegészítő sav-bázis katalitikus csoportként funkcionál, mely az -OH protonok elvonása révén elősegíti a szubsztrát koordinációját. Továbbá rámutattunk, hogy a tripodális szerkezet pozitívan befolyásolja a fémkomplexek szubsztrátkötő sajátságait, megnövelve ezzel a katalitikus hatékonyságot. A tripodális struktúra továbbá elősegíti többmagvú fémkomplexek kialakulását is, utat nyitva a nagyhatékonyságú kételektronos oxidációs mechanizmus felé: kétmagvú rendszereink még a nem peptid típusú pirokatechin oxidáz utánzó modellkomplexekkel összehasonlítva is kiemelkedő katalitikus aktivitást mutattak. Összegezve tehát, a munka során a tripodális peptidek egy új, hisztidintartalmú csoportját szintetizáltuk és tanulmányoztuk. Mélyreható, szisztematikus oldategyensúlyi és spektroszkópiai vizsgálatok segítségével feltártuk a koordinációs sajátságok hajtóerejét, és megállapítottuk a tripodális állványzat hatását a komplexek stabilitására, szerkezetére és katalitikus tulajdonságaira. Az eredményeink alátámasztják a tripodális alapvegyületnek a peptidszerű ligandumok fémkomplexeire gyakorolt jótékony hatását, és rámutat a tripodális peptidek alkalmazásában rejlő lehetőségekre az enzim-modellezés területén. (1) Nous avons voulu étudier l'effet de la plate-forme tripodale sur la stabilité thermodynamique des complexes métalliques formés.

RESUME FRANÇAIS

I. INTRODUCTION

(2) Nous avons voulu identifier le rôle du tripode et d'acide aminé dans la structure des espèces formées en analysant la géométrie, le nombre de coordination et les modes de liaison dominants.

(3) Nous avons voulu comparer les propriétés de coordination des ligands contenant de l'histidine en position N-ou C-terminale et étudier l'impact de la fonctionnalisation asymétrique sur la formation des complexes.

(4) Nous avons voulu tester l'activité catalytique des complexes métalliques formés dans de simples réactions modèle enzymatique, comme par exemple dans l'oxydation du catéchol ou dans la dismutation du superoxyde.

(

III. TECHNIQUES EXPERIMENTALES ET METHODES

Synthèses des ligands

Les ligands présentés dans ce travail ont été préparés et purifiés au laboratoire de synthèse du Département de Chimie Inorganique et Analytique de l'Université de Szeged.

Les ligands nta1his et nta1his-NH 2 ont été synthétisés en utilisant la méthode de synthèse peptidique en phase solide; tous les autres ligands ont été préparés par couplage peptidique en solution. Les structures schématiques des ligands synthétisés sont représentées sur la

Mesures cinétiques

Les activités catalytiques des complexes métalliques ont été étudiées dans des réactions d'hydrolyse du phosphoester, d'oxydation du catéchol et de dismutation du superoxyde. Les expériences ont été réalisées dans des solutions tamponnées, à une force ionique de 0,1 M et à 25°C. Les réactions ont été suivies par spectrophotométrie; la détermination des constantes de vitesse de la réaction du pseudo-premier ordre (k obs ) a été réalisée selon la méthode de la vitesse initiale. L'autohydrolyse ou le taux d'oxydation des substrats ont été pris en compte. Les paramètres cinétiques enzymatiques (k cat et K M ), utilisés pour la caractérisation des activités de catéchol oxydase, ont été calculés selon l'équation de Michaelis-Menten, avec une régression non linéaire des données.

IV. NOUVEAUX RESULTATS SCIENTIFIQUES

Figure 1 . 1 .

 11 Figure 1.1. Examples for basic tripodal ligands: tren (tris(2-aminoethyl)amine), nta (nitrilotriacetic acid) and tach (1,3,5-triaminocyclohexane).

Figure 1 . 1 .

 11 Figure 1.1.presents examples for some basic, extensively described and used tripodal ligands. The structure of the mentioned basic tripods, having one donor group per arm in a chelating position, normally allows tri-or tetradentate binding to a transition metal ion, and the free coordination sites can be occupied by solvent molecules or counter ions. With respect to geometry, the quality of the metal ion, the solvent and the flexibility of the ligand together determines coordination number and thus the structure of the complexes:

Figure 1 . 2 .

 12 Figure 1.2. Metal complex formation of tren in aqueous media.

Figure 1 . 3 .

 13 Figure 1.3. Examples for destabilization of multiimidazole-metal complexes (from previouswork in our laboratory[43]).

Figure 1 . 4 .

 14 Figure 1.4. Structure of copper(II) complexes in presence of (1) terminal His, (2) NH 2 -X-His, (3) NH 2 -X-X-His and (4) His-X-His type sequences.

Figure 1 . 5 .

 15 Figure 1.5. Dominant binding modes in terminal His-peptide complexes: CuL 2 (left) and CuH -1 L (right) complexes of His-Gly and Gly-His peptides, respectively (coordinating water molecules omitted).

Figure 1 . 6 .

 16 Figure 1.6. Left: Met form of the active center of catechol oxidase (COx) isolated from sweet potato (Ipomoea batatas) [48]; right: the reaction catalyzed by COx.



  Figure 1.7. Structures of ligand precursors of some highly active catecholase mimicking model complexes. Redrawn after (a) Das [71], (b) Meyer[67], (c) Klemm[69], (d) Casella[79], (e) Bouwman[68], (f) Reedijk[56] and co-workers.

Figure 1 . 8 .

 18 Figure 1.8. Schematic structure of the active center of Cu,Zn-SOD enzyme. Redrawn after Várnagy and co-workers [82].

Figure 1 . 9 .

 19 Figure 1.9. The model reaction used for SOD activity determination.

Figure 1 . 10 .

 110 Figure 1.10. Hydrolysis of DNA and RNA model compounds (BNPP: bis(4-nitrophenyl) phosphate, HPNP: 2-hydroxypropyl-p-nitrophenyl phosphate, NP: p-nitrophenol).

1 .

 1 Scheme 4.1. Schematic structure of the ligand tren3his (L 1 ).

L 1 HL 1 Figure 4 . 1 .

 1141 Figure 4.1. Speciation diagram (left) and table of formation and deprotonation constants (right) of the tren3his ligand system ([L 1 ] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K).

2 Figure 4 . 3 .

 243 Figure 4.3. Speciation diagrams of Cu(II)-L 1 1:1 (up) and 3:2 (down) systems ([L 1 ] tot = [Cu(II)] tot = 0.001 M and 1.5×[L 1 ] tot = [Cu(II)] tot = 0.001 M respectively, I = 0.1 M NaCl, T = 298 K).

1 BFigure 4 . 4 .Figure 4 . 5 .

 14445 Figure 4.4. pH-dependent UV-Vis spectra of Cu(II)-L 1 1:1 (A) and 3:2 (B) systems (A: [L 1 ] tot = [Cu(II)] tot = 0.0013 M, B: 1.5×[L 1 ] tot = [Cu(II)] tot = 0.0019 M, I = 0.1 M NaCl, T = 298 K). Corresponding calculated individual molar spectra can be found in Figure 4.8.

Figure 4 . 6 .

 46 Figure 4.6. pH-dependent EPR spectra of Cu(II)-L 1 1:1 (left) and 3:2 (right) system (0.95×[L 1 ] tot = [Cu(II)] tot = 0.0016 M and 1.4×[L 1 ] tot = [Cu(II)] tot = 0.0023 M respectively, I = 0.1 M NaCl, T = 298 K). Black: measured, red: simulated spectra.

Figure 4 . 7 .

 47 Figure 4.7. MALDI-TOF MS spectra measured in the Cu(II)-L 1 1:1 (left) and 3:2 (right) systems. Insert: Calculated spectra of the corresponding complexes ([C 24 H 38 N 13 O 3 Cu] + and [C 48 H 73 N 26 O 6 Cu 3 ] + , respectively).

Figure 4 . 8 .

 48 Figure 4.8. Individual molar UV-Vis spectra of mononuclear (A) and trinuclear (B) Cu(II)-L 1complexes.

Figure 4 . 9 .

 49 Figure 4.9. Individual molar CD spectra of mononuclear (A) and trinuclear (B) Cu(II)-L 1complexes.

2 . 2 (

 22 Scheme 4.2. Proposed schematic structure of copper(II)-L 1 complexes CuL 1 , CuH -2 L 1 and Cu 3 L 1 2

Figure 4 . 10 .

 410 Figure 4.10. Titration curves in Zn(II)-L 1 0:1 (white), 1:1 (gray) and 3:2 (black) systems (left); formation and deprotonation constants of zinc(II) complexes (right). Estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K.

1 Figure 4 . 12 .

 1412 Figure 4.12. Speciation diagrams of Zn(II)-L 1 1:1 (up) and 3:2 (down) systems; dotted lines represent the trinuclear species ([L 1 ] tot = [Zn(II)] tot = 0.001 M and 1.5×[L 1 ] tot = [Zn(II)] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 . 13 .

 413 Figure 4.13. Aromatic (A) and aliphatic (B) region of pH-dependent 1 H NMR spectra of Zn(II)-L 1 1:1 system (in H 2 O/D 2 O, [L 1 ] tot = [Zn(II)] tot = 0.0017 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 .

 4 Figure 4.14. 1 H NMR spectra of L 1 (A) and Zn(II)-L 1 1:1 (B,C) systems. pH = 7.2 (A), 7.3 (B) and 9.8 (C). (in H 2 O/D 2 O, [L 1 ] tot = 0.0023 M, I = 0.1 M NaCl, T = 298 K).

2 Scheme 4 . 3 .

 243 Scheme 4.3. Proposed structures of mono-and trinuclear Zn(II)-L 1 complexes.

Figure 4 . 15 .

 415 Figure 4.15. Aromatic region of the pH-dependent 1 H NMR spectra of Zn(II)-L 1 3:2 system (in H 2 O/D 2 O, [L 1 ] tot = 0.0023 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 .

 4 Figure 4.16. 1 H-1 H TOCSY NMR spectrum of Zn(II)-L 1 3:2 system at pH = 9.8 (in H 2 O/D 2 O, I = 0.1 M NaCl, T = 298 K).

  ). This observation supports the presence of several oligomers with different compositions. Applying the approximate rule (D ref /D x ) 3 = M x /M ref (where D and M stands for the diffusion coefficient and molar mass, ref. values: dioxane and acetonitrile), one can estimate several oligomers formed by the association of 2-8 trinuclear complexes (although uncertainties must be taken into account due to the non-spherical geometry and different hydration of the ligand and its Zn(II) complexes).

Figure 4 . 17 .Scheme 4 . 4 .

 41744 Figure 4.17. Aromatic region of 1 H NMR spectra in Zn(II)-L 1 3:2 system, and the measured diffusion coefficients by 1 H DOSY NMR. * indicates the C 2 H signal of the free ligand, the corresponding C 5 H signal at 6.85 ppm is not resolved (pH = 10, [L 1 ] tot = 0.0019 M, [Zn(II)] tot = 0.0027 M, I = 0.1 M NaCl, T = 298 K).

Scheme 4 . 5 .

 45 Scheme 4.5. Schematic structure of the ligand nta3his (L 2 ).

Figure 4 . 19 .

 419 Figure 4.19. pH-dependent 1 H NMR spectra of ligand nta3his ([L 2 ] tot = 0.0019 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 .L 2 .

 42 Figure 4.19. shows the 1 H NMR spectra recorded at different pH values in presence ofL2 . Both the nta CH 2 and the CH 2 protons are magnetically inequivalent, resulting (highly overlapping) AB-quartet signals in the whole pH range. The CH proton is not visible at any pH: it remains concealed by the water signal. This surprisingly high chemical shift is most probably due to the electron withdrawing effect of the neighboring amide NH group; similarly high (4.4-4.5 ppm) chemical shifts were observed for CH signal in Gly-His and Ala-His peptides[46,126]. The pH-dependent changes are in agreement with the potentiometrically observed deprotonation processes: the imidazole C 2 H, C 5 H and the amide NH proton signals shift considerably upfield between pH 6 and 9. Interestingly, the nta CH 2 signal also shifts during the deprotonation of the imidazole moiety; the increased electron density on these groups can be explained by a ceasing H-bond between the N tert and imidazolium groups. It is worth mentioning that the amide NH signal is visible almost up to pH 10, indicating unusually slow proton exchange processes for this group.

2 CuH 2 L 2 2 Cu 2 H -1 L 2 Cu 2 L 2 Cu 2 H -2 L 2 2 H -3 L 2 Figure 4 . 20 .

 222222222222420 Figure 4.20. Speciation diagrams of Cu(II)-L 2 1:1 (up) and 2:1 (down) systems ([L 2 ] tot = [Cu(II)] tot = 0.001 M and 2×[L 2 ] tot = [Cu(II)] tot = 0.001 M respectively, I = 0.1 M NaCl, T = 298 K).

Figure 4 . 21 . 2 Figure 4 . 22 .

 4212422 Figure 4.21. pH-dependent UV-Vis (left) and CD spectra (right) of Cu(II)-L 2 1:1 system ([L 2 ] tot = [Cu(II)] tot = 0.0015 M and 0.0016 M, respectively, I = 0.1 M NaCl, T = 298 K). UV-Vis insert: pH-dependent absorbances at 628 (♦) and 770 nm (□). CD insert: pH-dependence of  at 550 (∆), 640 (□) and 780 nm (♦).

  and 4.22 right).

Figure 4 .

 4 Figure 4.23. Anisotropic EPR spectra recorded at 77 K in Cu(II)-L 2 1:1 and 2:1 systems ([L 2 ] tot = [Cu(II)] tot = 0.0017 M and 1.9×[L 2 ] tot = [Cu(II)] tot = 0.0033 M, respectively, I = 0.1 M NaCl. Red: simulated, black: measured). Calculated EPR parameters of (a), (b) and (c) component spectra (related to the complexes CuH x L 2 (x = 2, 1, 0, -1), Cu 2 H x L 2 (x = -1, -2, -3) and CuH -2 L 2 , respectively) are listed in Table 4.3.

Figure 4 . 25 .

 425 Figure 4.25. pH-dependent UV-Vis (left) and CD (right) spectra of Cu(II)-L 2 2:1 systems (1.95×[L 2 ] tot = [Cu(II)] tot = 0.0029 M and 0.0031 M, respectively, I = 0.1 M NaCl, T = 298 K). UV-Vis insert: pH-dependent absorbances at 607 (♦) and 720 nm (□); CD insert: pH-dependent absorbances at 525 (□), 560 (▲) and 780 nm (♦).

2 Cu 2 H -1 L 2 Cu 2 H -2 L 2 Cu 2 H -3 L 2 Figure 4 . 26 .

 2222222426 Figure 4.26. Individual molar UV-Vis (left) and CD (right) spectra of dinuclear Cu(II)-L 2complexes, along with some difference spectra.The following deprotonation (pK Cu 2 H -1 L 2 = 6.65) results in ~ 60 nm blue shift of the d-d maximum and important change on the CD spectra, which indicates amide nitrogen coordination to the second metal ion (the Gly-His-like coordination in the mononuclear complexes is unchanged up to pH 9). Indeed, the visible difference spectra between Cu 2 H -2 L 2 and CuH -1 L 2 ( d-d max ~ 585 nm, Figure4.26.) is again in accordance with a {2N im ,N  ,COO  } type coordination[START_REF] Prenesti | [END_REF] in the equatorial plane of the second copper(II) (Scheme 4.6). This species has surprisingly high stability: it is the dominant complex over nearly 3 pH units. The next deprotonation (pK Cu 2 H -2 L 2 = 9.35) induces further characteristic changes on CD spectra both in the UV and visible region (Figure4.26), which reflects the metal coordination of the third amide nitrogen. On the other hand, the visible spectrum shows ~ 20 nm red shift between pH 8-10.3 (Figure4.25). The spectral changes exclude the formation of a binding mode present in CuH 2 L2 . The third amide nitrogen binds most likely to the second metal ion, creating a {N tert ,N  ,N im ,O; 2N  ,2N im } coordination mode in Cu 2 H -3 L2 . Peptides with -HXH-sequence undergo two strongly overlapping amide deprotonations, due to the preferred formation of a {2N  ,2N im } coordinated species[65,91]. However, in the present case the two successive amide deprotonations are well separated, because they are involved in a considerably less favored, eight-membered chelate ring. Moreover, this chelate ring contains the metal-bound tertiary amine, too, which probably makes the conformational change needed for the Cu 2 H -2 L 2  Cu 2 H -3 L 2 + H + deprotonation more difficult, resulting in the dominant formation of Cu 2 H -2 L 2 at pH 8.

2 Cu 2 H -2 L 2 Scheme 4 . 6 .

 22246 Scheme 4.6. Proposed schematic structure of copper(II)-L 2 complexes CuH -1 L 2 , CuH -2 L 2 and Cu 2 H -2 L 2 .

2 Figure 4 . 27 .

 2427 Figure 4.27. Speciation diagrams of Zn(II)-L 2 at 1:3.5 (up) and 1:1 (down) metal-to-ligand ratios ([L 2 ] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K). Dashed lines represent unbound ligand species; gray field indicates pH region of precipitation.

2 Figure 4 . 28 .

 2428 Figure 4.28. pH-dependent 1 H NMR spectra of Zn(II)-L 2 1:1 system ([L 2 ] tot = 0.0029 M, I = 0.1 M NaCl, T = 298 K).

5 Figure 4 . 29 . 1 H

 54291 Figure 4.29. 1 H NMR spectra of Zn(II)-L 2 system recorded around pH 10 (up) and 8 (down), at elevating Zn(II)-to-ligand ratios. ([L 2 ] tot = 0.0012 M, I = 0.1 M NaCl, T = 298 K).

7 .

 7 Scheme 4.7. Schematic structures of the ligands nta1his and nta1his-NH 2 .

7

 7

  and A.8).

Table 4 . 6 .

 46 Formation and deprotonation constants of copper(II)-and zinc(II)-nta1his-NH 2 complexes (estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K).

ZnH - 2 LFigure 4 . 30 .

 2430 Figure 4.30. Speciation diagrams in Cu(II)-nta1his-NH 2 1:1 (left) and Zn(II)-nta1his-NH 2 1:1 (right) systems ([L] tot = 0.001 M, [Cu(II)] tot = [Zn(II)] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 . 31 .

 431 Figure 4.31. Individual molar UV-Vis spectra of the main Cu(II)-nta1his-NH 2 complexes.

Scheme 4 . 8 .

 48 Scheme 4.8. Schematic structures of the ligands tren2his(L 3 ) and tren1his(L 4 ).

  and 4.33): the C 2 H, C5 H and CH proton signals present significant displacement in the relevant pH ranges. The pK values of tren-like primary and NH 2 amino nitrogens are between pK = 9.04-9.90 and 6.82-7.69, respectively. Taking into account the tris-histidyl analog tren3his as well (Section 4.1.1.), the increasing overall positive charge of the ligands results in increased acidity of the imidazolium rings (pK = 3.87-5.41) in the trenXhis (X = 1, 2, 3) series.

Figure 4 . 33 . 1 H

 4331 Figure 4.33. 1 H NMR titration of ligand L 4 (in H 2 O, [L 4 ] tot = 0.0017 M, I = 0.1 M NaCl, T = 298 K).

3 Cu 2 H -1 L 3 Cu 2 H -2 L 3 Cu 2 H -3 L 3 Figure 4 . 34 .

 3232323434 Figure 4.34. Distribution of the species in Cu(II)-L 3 1:1 (up) and 2:1 (down) systems ([L 3 ] tot = 0.001 M, [Cu(II)] tot = 0.001 M and 0.002 M, respectively, I = 0.1 M NaCl, T = 298 K). Secondary axis: measured CD intensities at 325 nm (up: green, down: blue) and 675 nm (orange).

Figure 4 .

 4 36. A) indicate rearrangement of the coordination sphere. The changes of CD spectra (Figure 4.36. B) support different coordination mode of the histidyl unit(s) in CuHL 3 and CuH -1 L 3 . The important blue shift of the copper(II) d-d band (635  555 nm, Figure 4.36.

Figure 4 . 36 .

 436 Figure 4.36. Individual molar UV-Vis (A) and CD (B) spectra of the main species in Cu(II)-L 3 1:1 system. Black dashed line corresponds to difference spectrum.

Figure 4 . 37 . 2 9 .

 43729 Figure 4.37. Individual molar UV-Vis (A) and CD (B) spectra of the main species in Cu(II)-L 3 2:1 system.

Table 4 . 9 .

 49 Formation and deprotonation constants of zinc(II)-L 3 complexes (estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K).

3 ZnL 3 ZnH -1 L 3 ZnH -2 L 3 Zn 2 3 Zn 2 H -3 L 3 Zn 2 H -2 L 3 3 ZnH 2 L 3 3 Figure 4 . 38 .

 33332323233233438 Figure 4.38. Speciation diagrams in Zn(II)-L 3 1:1 (up) and 2:1 (down) systems ([L 3 ] tot = 0.001 M, [Zn(II)] tot = 0.001 and 0.002 M, I = 0.1 M NaCl, T = 298 K), dashed lines represent unbound ligand (H x L 3 ) species.

Figure 4 . 39 . 1 H

 4391 Figure 4.39. 1 H NMR titration of Zn(II)-L 3 1:1 system (in H 2 O, [L 3 ] tot = [Zn(II)] tot = 0.0017 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 .Figure 4 . 40 . 1 H

 44401 Figure 4.40. 1 H NMR spectra of L 3 (A), Zn(II)-L 3 1:1 (B,D,E) and 2:1 (C,F, and G after deuteration of im-C 2 group) systems ([L 3 ] tot = 0.0018 M, I = 0.1 M NaCl, T = 298 K). Marked* signal corresponds to dioxane reference at 3.70 ppm. At metal excess, the evaluation of potentiometric data indicated the formation of three dinuclear complexes with compositions of Zn 2 H -2 L 3 , Zn 2 H -3 L 3 and Zn 2 H -4 L 3 . Their formation starts around pH 7 (Figure 4.38. down), which is in good agreement with the 1 H NMR spectra recorded at twofold metal excess: the spectral features are identical to that observed at Zn(II)-L 3 1:1 ratio until pH 7 (Figure 4.40. B, C). Starting from neutral pH, several CH proton signals can be seen, and more importantly, six sharp signals appear in the aromatic region of 1 H NMR spectrum (Figures 4.40. F and 4.41). The latter indicates three chemically different environments for the imidazole rings with nearly equal populations in the slow exchanging oligonuclear complexes. 2D TOCSY measurements and 1 H NMR spectrum of the complex(es) deuterated at the imidazole C 2 carbon (Figure 4.40. G) allowed the identification of the C 2 H/C 5 H pairs. Accordingly, at pH 8.2 the C 2 H signals are strongly upfield-shifted by 0.29, 0.46 and 1.05 ppm in the slow exchanging oligonuclear complexes, as compared to the free ligand signals (Figure 4.41. right).

Figure 4 . 41 . 3 Scheme 4 . 10 .

 4413410 Figure 4.41. Aromatic region of 1 H NMR spectra of Zn(II)-L 3 system. Left: pH dependence at Zn(II)-L 3 1.9:1 ratio; right: dependence on the Zn(II)-L 3 ratios at pH 8.2 (in H 2 O, [L 3 ] tot = 0.0018 M, I = 0.1 M NaCl, T = 298 K). Marked signals demonstrate corresponding C 2 H (black symbols) and C 5 H (empty symbols) protons.

  2-0.4 ppm upfield shift of the imidazole and CH signals. In the present system, the wide range of chemical shifts of imidazole and CH signals upon metal ion binding (Figures 4.40. F and 4.41.) indicates the presence of both amide-and imidazolate-bound zinc centers.

  2 H -2 L 3 = (Zn 2 H -2 L 3 ) 2 , through the formation of two imidazolato-bridges and the re-protonation of two zinc-bound amide nitrogens. The proposed structures for Zn 2 H -2 L 3 and (Zn 2 H -2 L 3 ) 2 are depicted in Figure 4.42.

Figure 4 . 42 .

 442 Figure 4.42. Proposed structures for Zn 2 H -2 L 3 and (Zn 2 H -2 L 3 ) 2 .The proposed dimerization involves simple proton transfer from the imidazole N 1 H to the deprotonated amide nitrogen. During this process, the imidazole C 2 H protons become close to the tren CH 2 groups of the other ligand molecule in space. Accordingly, we observed ROESY correlation between two imidazole C 2 H protons (Figure4.43), tentatively assigned to (Zn 2 H -2 L 3 ) 2 , and two aliphatic signal at 2.58 and 2.78 ppm, most likely corresponding to the inequivalent protons of N tert -CH 2 groups. The simultaneous presence of the two complexes (Zn 2 H -2 L 3 and (Zn 2 H -2 L 3 ) 2 ) is probably the simplest way to explain the six sharp signals observed in the aromatic region of the NMR spectra at 2:1 Zn(II)-L 3 ratio.

Figure 4 . 43 .

 443 Figure 4.43. 2D ROESY (green) and TOCSY (red) spectra in Zn(II)-L 3 2:1 system at pH 8.2.

4 Figure 4 . 44 .

 4444 Figure 4.44. Distribution of the species in Cu(II)-L 4 1:1 system ([L 4 ] tot = [Cu(II)] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K). Secondary axis: measured CD intensities at 305 nm (red), 535 nm (green) and 635 nm (blue).

4 BFigure 4 . 45 .

 4445 Figure 4.45. Individual molar UV-Vis (A) and CD (B) spectra of the forming species in Cu(II)-L 4 1:1 system.

Figure 4 . 46 . 13 C

 44613 Figure 4.46. 13 C NMR spectra of L 4 in presence of 0% (black), 5% (red) and 10% (blue) Cu(II) (in H 2 O, pH 8.5, [L 4 ] tot = 0.05 M, T = 298 K). Axis is scaled to the spectrum of the free ligand.Obviously, in an alternative structure NH 2 may replace the imidazole ring. In order to support one of the above mentioned binding modes, we recorded 1 H and13 C NMR spectra of L 4 in the presence of copper(II). On the 1 H NMR spectra the paramagnetic copper(II) induced only general, not selective broadening of the signals. On the other hand, the 13 C NMR spectra indicated notable broadening of the signals corresponding to the imidazole moiety (C 2 H, C 4 , Figure 4.46.) as compared to the minor broadening of the CH signal. This observation suggests the participation of imidazole ring in the coordination in CuH -1 L 4 (Scheme 4.11).

4 ZnH 2 L 4 4 ZnH -2 L 4 Figure 4 . 47 .

 42444447 Figure 4.47. Speciation diagrams in Zn(II)-L 4 1:1 system ([L 4 ] tot = [Zn(II)] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K).

Figure 4 . 48 .

 448 Figure 4.48. pH-dependent 1 H NMR spectra of Zn(II)-L 4 1:1 system (A,B), 1:1.5 system (D) and comparison with the spectra of free ligand (C,E). In H 2 O, dioxane reference (3.70 ppm), [L 4 ] tot = 0.0016 M, I = 0.1 M NaCl, T = 298 K.

12 .

 12 Scheme 4.12. Proposed structure of the main Zn(II)-L 4 complexes.

Figure 4 . 49 .

 449 Figure 4.49. Simulated predominance diagrams in the Cu(II)-(left) and Zn(II)-(right) L 1 -L 3 -L 4 1:1:1:1 quaternary systems. [Cu(II)] tot = [Zn(II)] tot = [L 1 ] tot = [L 3 ] tot = [L 4 ] tot = 0.001 M.

Figure 4 . 50 .

 450 Figure 4.50. Simulated predominance diagrams in the Cu(II)-L 1 -L 2 1:1:1 ternary system. [Cu(II)] tot = [L 1 ] tot = [L 2 ] tot = 0.001 M.

Figure 4 . 51 .

 451 Figure 4.51. Simulated predominance diagrams in the Cu(II)/Zn(II)-L 1 -histamine 1:1:3 (left) and Cu(II)-L 2 -Gly-His 1:1:1 (right) ternary systems. [Cu(II)] tot = [Zn(II)] tot = [L 1 ] tot = 0.001 M, [histamine] tot = 0.003 M, [Cu(II)] tot = [L 2 ] tot = [Gly-His] tot = 0.001 M.

Figure 4 . 52 .

 452 Figure 4.52. Simulated predominance diagrams in the Cu(II)-L 1 -L 3 -L 4 -GGH 1:1:1:1:1 system. [Cu(II)] tot = [L 1 ] tot = [L 3 ] tot = [L 4 ] tot = [GGH] tot = 0.001 M.

Figure 4 . 53 .

 453 Figure 4.53. pH-dependent reaction rate constants (k obs,corr , secondary axis) in the investigated Cu(II)-L 1 3:2 (A), Cu(II)-L 2 2:1 (B), Cu(II)-L 3 1:1 (C) and Cu(II)-L 3 2:1 (D) systems, with the corresponding speciation diagrams (primary axis). Applied concentrations in Table4.12.

Figure 4 . 54 .

 454 Figure 4.54. Dependence of the initial reaction rates on H 2 DTBC concentration promoted by Cu(II)-L systems (♦: Cu(II)-L 1 3:2, pH corr 7.8, □: Cu(II)-L 2 2:1, pH corr 8.9, ∆: Cu(II)-L 3 1:1, pH corr 7.4, : Cu(II)-L 3 2:1, pH corr 8.7); applied concentrations in Table4.12. Solid lines correspond to

  ol b ligand B3: 3-(3'-methyl-2'-pyridylimino)isoindoline-1-one (MePyOIND) c ligand B4: 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (BPMP) d ligand B5: naproxen (nap) and 3-picoline (3-pic) e ligand B6: the copper(II) complex is referred as bis[1,2-O-isopropylidene-6-N-(3-acetylbut-3-en-2-one)amino-6deoxyglucofuranoso]dicopper (H 2 3a) f ligand B7: L 1* SSL 1* g ligand B8: a pyrazolate-derived compartmental ligand bearing pyridyl arms (HL 2 ) h ligand B9: 2,6-bis(R-iminomethyl)-4-methyl-phenolato type Schiff-base ligand (H 2 L2 2 ) i ligand B10: 2,6-bis(R-iminomethyl)-4-methyl-phenolato type Schiff-base ligand (L2 1 ) j native enzyme isolated from Ipomoea batatas; kinetic parameters reported for catechol substrate

Figure 4 . 55 .

 455 Figure 4.55. Dependence of reaction rate constants (k obs,corr ) on complex concentrations in Cu(II)-L 1 3:2 (A, pH 7.8), Cu(II)-L 2 2:1 (B, pH 8.9) and Cu(II)-L 3 2:1 systems (C, pH 8.7); applied concentrations in Table4.12.

Figure 4 . 56 .

 456 Figure 4.56. Dependence of reaction rate constants (k obs, ) on oxygen concentrations in Cu(II)-L 13:2 (A, pH 7.8), Cu(II)-L 2 2:1 (B, pH 8.9) and Cu(II)-L 3 2:1 systems (C, pH 8.7); applied concentrations in Table4.12.

Figure 4 Figure 4 . 57 .

 4457 Figure 4.57. pH-dependent absorption at 434 nm at different Cu(II)-L 1 -H 2 4NC ratios: × 0:0:1,  3:2:1, □ 3:2:2, ∆ 3:2:3 ([H 2 4NC] tot = 2.5×10 -4 M, in EtOH/H 2 O 50/50%). Insert: Individual molar spectra of differently protonated 4NC species.

Figure 4 . 58 .

 458 Figure 4.58. H 2 DTBC-dependent UV-Vis spectra of Cu(II)-L 1 3:2 system under anaerobic conditions ([complex] tot = 2.4×10 -4 M, in EtOH/H 2 O 50/50%, pH 7.8). Insert (left): UV region of the spectra; (right): absorbance at 440 nm in function of eq. H 2 DTBC in presence (■) and in absence (□) of Cu(II)-L 1 3:2 system (solid line is fitted curve, see text).

Figure 4 . 59 .

 459 Figure 4.59. UV-Vis (A) and CD spectra (B) of the Cu(II)-L 3 2:1 system recorded under inert (argon) conditions, upon addition of 0-20 eq. H 2 DTBC (in EtOH/H 2 O 50/50%, pH corr = 8.7, [complex] tot = 5.3×10 -4 M). Insert: CD intensities in function of eq. H 2 DTBC (solid line is fitted curve, see text).

Figure 4 . 60 .

 460 Figure 4.60. H 2 DTBC-dependent UV-Vis spectra of Cu(II)-L 2 2:1 system under anaerobic conditions ([complex] tot = 2.4×10 -4 M, in EtOH/H 2 O 50/50%, pH 8.9). Insert: UV region of the measured spectra.

Figure 4 . 61 .

 461 Figure 4.61. H 2 O 2 ( and □) and DTBQ ( and ) formation in presence of Cu(II)-L 1 3:2 (A) and Cu(II)-L 2 2:1 systems (B) (pH 7.8 and 8.5, [complex] tot = 5×10 -5 M, [H 2 DTBC] o = 2×10 -3 M, I = 0.1 M).

10 GaussFigure 4 . 62 .

 10462 Figure 4.62. EPR spectrum of the 3,5-di-tert-butyl-semiquinone radical under aerobic reaction conditions, in presence of Cu(II)-L 1 3:2 complex (in EtOH/H 2 O 50/50, [complex] tot = 5×10 -5 M, [H 2 DTBC] o = 2×10 -3 M, pH = 8.0). Acquisition settings: 0.84 G modulation amplitude, 5×10 4 receiver gain, 16 scans, 5 mW microwave power, 50 G scan width.

14 .

 14 Scheme 4.14. Linear peptide sequences studied in catechol oxidation.

Figure 4 . 63 .Figure 4 . 64 .

 463464 Figure 4.63. pH-dependent reaction rate constants (k obs,corr = k obsk obs,auto ) observed in equimolar Cu(II)-peptide systems, in 50/50% EtOH/H 2 O buffered (HEPES, CHES) solutions, [Cu(II)] tot = [P] tot = 5.0×10 -5 M, T = 298 K, I = 0.1 M. Individual conditions: [H 2 DTBC] o = 3×10 -3 M for Cu(II)-P1, -P2 and -P3, [H 2 DTBC] o = 8×10 -4 M, for Cu(II)-P4 and -P5 systems.

Scheme 4 . 15 .

 415 Scheme 4.15. Proposed mechanism of catalytic oxidation of 3,5-di-tert-butylcatechol by amidecoordinated copper(II) complexes of multihistidine peptides.

Figure 4 . 65 .

 465 Figure 4.65. Superoxide dismutase activity of Cu(II)-L 1 1:1 (A), Cu(II)-L 2 2:1 (B), Cu(II)-L 3 1:1 (C, ♦) and 2:1 (C, □) systems at pH 7.4.

( 7 )

 7 Tripodális peptidkomplexeinket, valamint néhány lineáris peptid-réz(II)komplexet szuperoxid dizmutáz funkcióban is teszteltük. A Cu(II)-L 4 rendszeren kívül az összes réz(II)-ligandum rendszer jelentős SOD-aktivitással rendelkezik az alkalmazott McCord-Fridovich indirekt tesztreakcióban. A SOD-utánzó komplexek IC 50 értékei mind előnyösebbek a szabad réz(II)ionra meghatározott értéknél, és más, korábban vizsgált hisztidintartalmú peptidkomplexek értékeihez hasonlítanak. Aktivitásuk azonban közel két nagyságrenddel alulmarad a natív Cu,Zn-SOD hatékonyságához képest.

Figure I. 1 .Figure I. 2 .

 12 Figure I.1. Déstabilisation de l'environment multi-imidazolique en complexes de cuivre(II) et zinc(II) (du travail précédent de nos laboratoires).

Figure 2 Figure II. 1 .

 21 Figure II.1.

1 . 1 . 2 . 2 Figure IV. 1 . 2 (

 112212 Figure IV.1. Distribution des espèces dans les systèmes Cu(II)-L 1 1:1 (gauche) et 3:2 (droite); [L 1 ] tot = [Cu(II)] tot = 0.001 M et 1.5×[L 1 ] tot = [Cu(II)] tot = 0.001 M, respectivement, I = 0.1 M NaCl, T = 298 K.

Figure IV. 2 .Figure IV. 3 .

 23 Figure IV.2. Spectres de masse MALDI-TOF mesuré dans les systèmes Cu(II)-L 1 1:1 (gauche) et 3:2 (droite). Insertion : spectres calculés pour les complexes correspondants ([C 24 H 38 N 13 O 3 Cu] + et [C 48 H 73 N 26 O 6 Cu 3 ] + , respectivement).

2 Figure IV. 4 .

 24 Figure IV.4. Spectres CD individuels calculés pour les complexes Cu(II)-L 1 mononucléaires (A) et trinucléaires (B).

1 Figure IV. 5 .

 15 Figure IV.5. Distribution des espèces dans les systèmes Zn(II)-L 1 1:1 (gauche) et 3:2 (droite); [L 1 ] tot = [Zn(II)] tot = 0.001 M et 1.5×[L 1 ] tot = [Zn(II)] tot = 0.001 M, respectivement, I = 0.1 M NaCl, T = 298 K.

Figure IV. 6 .

 6 Figure IV.6. Haut : Région aromatique des spectres 1 H NMR en fonction du pH dans le système Zn(II)-L 1 3:2 (en H 2 O/D 2 O, [L 1 ] tot = 0.0023 M, I = 0.1 M NaCl, T = 298 K). Bas : Coefficients de diffusion mesurés par 1 H DOSY NMR. * indique le signal C 2 H du ligand libre ; le signal C 5 H correspondant est mal résolu (pH = 10, [L 1 ] tot = 0.0019 M, [Zn(II)] tot = 0.0027 M, I = 0.1 M NaCl, T = 298 K).

6 Figure IV. 7 . 2 . 2 . 2 .

 67222 Figure IV.7. Structuration proposée pour la formation des espèces oligomères dans le système Zn(II)-L 1 3:2.

2 CuH 2 L 2 2 Cu 2 H -1 L 2 Cu 2 L 2 Cu 2 H -2 L 2 2 H -3 L 2 Figure IV. 8 . 1 ( 2 Figure IV. 9 . 2 Cu 2 H -2 L 2 Figure IV. 10 . 2 :CuH - 1 L 2 , 2 . 2 . 3 . 2 Figure IV. 11 . 5 Figure IV. 12 . 3 . 2 3. 1 . 3 . 2 .

 22222222222281292221021222321151232132 Figure IV.8. Distribution des espèces dans les systèmes Cu(II)-L 2 1:1 (gauche) et 2:1 (droite); [L 2 ] tot = [Cu(II)] tot = 0.001 M et 2×[L 2 ] tot = [Cu(II)] tot = 0.001 M, respectivement, I = 0.1 M NaCl, T = 298 K.

ZnH - 2 LFigure IV. 13 .

 213 Figure IV.13. Distribution des espèces dans le système Cu(II)-nta1his-NH 2 1:1 (gauche) et Zn(II)-nta1his-NH 2 1:1 (droite) ; [L] tot = 0.001 M, [Cu(II)] tot = [Zn(II)] tot = 0.001 M, I = 0.1 M NaCl, T = 298 K.

Figure IV. 14 . 4 . 4 . 1 . 3 Cu 2 H -1 L 3 Cu 2 H -2 L 3 Cu 2 H -3 L 3 Figure IV. 15 . 3 ZnL 3 ZnH -1 L 3 ZnH -2 L 3 Zn 2 3 Zn 2 H -3 L 3 Zn 2 H -2 L 3 3 ZnH 2 L 3 3 Figure IV. 16 .Figure IV. 17 . 3 . 4 . 3 . 3 Figure IV. 18 . 3 . 5 . 5 . 1 . 4 ZnH 2 L 4 4 ZnH -2 L 4 Figure IV. 19 .

 1444132323231533332323233233161734331835514244419 Figure IV.14. Spectres UV-Vis individuels calculés pour les complexes majeurs de Cu(II)-nta1his-NH 2 .

5. 2 .Figure IV. 20 . 4 :CuH 2 L 4 , 4 . 6 .

 22042446 Figure IV.20. Structures proposées pour les complexes principaux de cuivre(II)-L 4 : CuH 2 L 4 , CuHL 4 et CuH -1 L 4 .

Figure IV. 21 .

 21 Figure IV.21. Les vitesses initiales de l'oxydation du H 2 DTBC en fonction de la concentration du substrat dans les systèmes catalytiques Cu(II)-L (♦: Cu(II)-L 1 3:2, pH corr 7.8, □: Cu(II)-L 2 2:1, pH corr 8.9, ∆: Cu(II)-L 3 1:1, pH corr 7.4, : Cu(II)-L 3 2:1,

Figure IV. 22 . 2 . 7 .

 2227 Figure IV.22. Mécanisme proposé pour l'oxydation de 3,5-di-tert-butylcatéchol catalysée par les systèmes cuivre(II)-L 1 et -L 2 .

Table 1 . 1 .

 11 Stability constants of ML complexes of tren

  at I = 0.1 M, T = 25 C or 20 C*.

	M(II)	logK ML / L = tren	logK ML / L = nta	logK ML / L = EDTA
	Cu(II)	18.5	12.96*	18.86*
	Zn(II)	14.5	10.66*	16.68*
	Mn(II)	5.77	7.44*	13.95*
	Fe(II)	8.8	8.83*	14.33*
	Fe(III)		16.26*	25.1*
	Co(II)	12.69	10.4*	16.49*
	Co(III)			41.5
	Ni(II)	14.6	11.54*	18.52

Table 1 . 2 .

 12 Superoxide dismutase activity of some copper(II)-ligand complexes.

	Cu(II)-ligand systems	IC 50 (M)	references	k (M -1 s -1 )
	Cu,Zn-SOD (pH 6.8)	0.0045	[65]	3.3×10 8
	Cu(II)-Ac-HSarHSarHSarH-NH 2 1:1 (pH 7.4)	0.0442	[82]	3.4×10 7
	Cu(II)-His-BIMA 3:2 (pH 7.4)	0.070	[85]	2.1×10 7
	Cu(II)-Ac-HGHG-OH 1:1 (pH 7.4)	0.083	[86]	3.6×10 7
	Cu(II)-HAHPH-NH 2 1:1 (pH 6.8)	0.0841	[82]	1.8×10 7
	Cu(II)-apz-pn 1:1 (pH 7.4)	0.0902	[87]	[NBT] not reported
	Cu(II)-HADHDHKK-NH 2 1:1 (pH 7.0)	0.11	[88]	1.4×10 7
	Cu(II)-Ac-(PHGGGWGQ) 4 -NH 2 1:1 (pH 6.6)	0.12	[89]	1.3×10 7
	Cu(II)-Ac-HHGH-OH; 1:1 (pH 7.5)	0.15	[65]	1.0×10 7
	Cu(II)-HGDHMHNHDTK-OH 1:1 (pH 7.0)	0.19	[90]	7.9×10 6
	Cu(II)-Ac-HVH-NH 2 ; 1:1 (pH 7.4)	0.20	[91]	7.5×10 6
	Cu(II)-HGHG-OH 1:1 (pH 7.4)	0.67	[86]	4.5×10 6
	Cu(HPO 4 ) (pH 7.4)	1.06	[91]	6.2×10 6

  H NMR (500 MHz, D 2 O/H 2 O, pH 7.19)  (ppm): 2.46 (m, 6H, N tert -CH 2 ), 2.98 (m, 6H, CH 2 ), 3.18 (m, 6H, CH 2 -NHCO), 3.88 (t, 3H, J = 7.0 Hz, CH), 6.96 (s, 3H, im C 5 H), 7.74 (s, 3H, im C 2 H). ESI-MS m/z calcd for C 24 H 40 N 13 O 3 [M+H] + 558.34, found 557.97 (z = 1) and [M+2H] 2+ 278.98 (z = 2).

  H NMR (500 MHz, D 2 O/H 2 O, pH 7.08)  (ppm): 2.95 (dd, 3H, J AB = 15.3 Hz, J HH = 4.5 Hz, CH 2 ), 2.96 (d, 3H, J AB = 15.3 Hz, N tert -CH 2 ), 3.06 (d, 3H, J AB = 15.3 Hz, N tert -CH 2 ), 3.16 (dd, 3H, J AB = 15.3 Hz, J HH = 4.5 Hz, CH 2 ), 7.00 (s, 3H, im C 5 H),

	7.99 (s, 3H, im C 2 H), 8.30 (d, 3H, CONH). ESI-MS m/z calcd for C 24 H 31 N 10 O 9 [M + H] +
	603.23, found 603.2 (z = 1) and [M+2H] 2+ 302.0 (z = 2).

  Table of the observed reaction rate constants and related discussion can be found in Appendix section A.26. and Table A.2.

Table 4 . 1 .

 41 Formation constants and spectroscopic (UV-Vis, CD and EPR) parameters of copper(II)-L 1 complexes (estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K).

	Cu p H q L r	log pqr	a	 d-d max (nm),  (M -1 cm -1 )	 CD  (M -1 cm -1 ) max (nm),	g o	A o (G)
	131	29.32(1) a [29.21(3)] b	650, 56.3	630, 0.41 312, -0.42	2.136(2)	61(1)
	121	25.66(1) a [25.54(4)] b	602, 99.9	624, 0.96 318, -0.93	2.109(6)	71.3(7)
	111	20.11(1) a [19.95(9)] b	614, 107.7	654, 0.76 318, -0.77	2.112(6)	68.7(6)
	101	13.19(1) a [13.18(9)] b	638, 100.5	690, 0.65 318, -0.55	2.111(6)	59.5(6)
	1-11	4.07(2) a [4.01(6)] b	626, 155.0	715, 0.64 612, -0.15	2.106(3)	9(5)
	1-21	-6.80(2) a [-6.84(5)] b	596, 200.8	708, -0.62 312, 1.64 582, 0.60	2.089(3)	63(3)
	322	49.57(2) a [49.60(4)] b	626, 189.0	636, 1.82 318, -1.82	2.102(3) c 2.133(2) c	64.1(7) c 48.7(6) c
	302	38.20(3) a,d	630, 198.2	678, 1.57 318, -1.30	2.103(2)	57(3)
					702, 1.46
	3-12	30.93(4) a,d	638, 264.7	546, 0.30	2.124(3)	60(2)
					312, -1.09
	3-22	22.73(4) a,d	632, 262.2	708, 1.92 594, -0.49	2.106(4)	61(3)
					714, 1.57
	3-32	14.03(4) a,d	616, 304.3	612, -0.05	2.099(2)	51(4)
					312, 0.42
					690, -0.88
	3-42	3.77(4) a,d	614, 329.5	576, 1.57 318, 5.20	2.107(3)	52(3)
					702, -0.38
	3-52	-6.99(3) a,d	610, 320.5	582, 1.57	2.097(3)	56
					318, 4.40

  2 L 1 ( d-d max = 602 nm, g o = 2.109, A o = 71.3 G, Table 4.1.) indicate considerably increased ligand field strength as compared to CuH 3 L 1 , which supports the coordination of four nitrogens in CuH 2 L 1 , thus a {NH 2 ,3N im } binding mode in the equatorial plane of copper(II). The copper(II) promoted deprotonation of the second amino nitrogen of L 1 should lead to the highly stable bis-histamine-like {2NH 2 ,2N im } coordination in the equatorial plane of CuHL 1 . However, the slight red shift of the d-d transitions and the decrease of A o of this species ( d-d max = 614 nm, g o = 2.112, A o = 68.7 G, Table 4.1.) as compared to CuH 2 L 1 , indicate some distortion of the crystal field around the metal ion. Considering that five N-donors are available for metal ion binding in CuHL 1 , this distortion is most likely caused by the axial coordination of the third imidazole nitrogen {2NH 2 ,2N im +N im }. During the next copper(II) promoted deprotonation (CuHL 1  CuL 1 ) an even more important red shift of the visible/CD spectra and decrease of A o was observed ( d-d max = 638 nm, g o = 2.111, A o = 59.5 G, Table

  1 ({2NH 2 ,2N im +NH 2 }, Scheme 4.2).

	pH	pH
	11.30
		11.00
	10.73
		10.15
	10.18
	9.68	9.44
	8.98	8.47
	8.23	7.59
	7.44	7.09
	6.85	6.24
	6.18	5.60
	5.40	5.14
	4.55	4.62
	3.90	3.82
	3.34
		2.96
	2.77	2.43
	3000 3200 3400 3600	3000 3200 3400 3600
	Magnetic field [G]	Magnetic field [G]

  .7). The obtained isotopic distribution patterns are in very good agreement with those calculated for the molecular ions [CuH -1 L 1 ] + (=[C 24 H 38 N 13 O 3 Cu] + ) and [Cu 3 H -5 L 1 2 ] + (=[C 48 H 73 N 26 O 6 Cu 3 ] +

  .1). All trinuclear complexes have detectable EPR spectra (Figures 4.6. and A.22), indicating weakly interacting metal ions. Nevertheless, only the spectra of Cu 3 H 2 L

1405.34 1401.32 1401.34 1405.30 Figure 4.11.

  .11). The detected m/z values and isotopic distribution patterns are in very good agreement with the calculated spectra for [ZnH -1 L 1 ] + (= [C 24 H 38 N 13 O 3 Zn] + ) and [Zn 3 H -4 L 1 2 (ClO 4 )] + (= [C 48 H 74 N 26 O 10 ClZn 3 ] + ). MALDI-TOF MS spectra measured in the Zn(II)-L 1 1:1 (left) and 3:2 (right) systems. Insert: Calculated spectra of the corresponding complexes ([C 24 H 38 N 13 O 3 Zn] + and [C 48 H 74 N 26 O 10 ClZn 3 ] + ). The monocomplexes ZnH 3 L 1 and ZnH 2 L 1 are formed around pH 4-5 in a strongly The selective broadening of 1 H NMR signals of the imidazole protons (C 2 H, C 5 H) around pH 5 (Figure 4.13.) indicates imidazole coordination in these species.

	overlapped manner.
										Zn p H q L r	log  pqr	pK
										131		24.76(2) 4.90
										121		19.86(1) 5.42
										111		14.44(1) 6.49
										101		7.95(1)	8.10
										1-11		-0.15(2) 10.24
										1-21		-10.39(3)
										302		22.87(4)
										3-22		9.62(3)	8.04
										3-32		1.58(4)	8.90
										3-42		-7.32(4)
		620.22			620.24
			615	620	625	630	635
						m/z
													1400	1405	1410	1415
														m/z
	615	620	625	m/z		630		635	1400	1405	1410	m/z	1415	1420	1425

The distribution of the differently protonated complexes is presented on Figure

4

.12.

Table 4 . 2 .

 42 Formation constants and spectroscopic (UV-Vis and CD) parameters of copper(II)-L 2 complexes (estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K).

		log	 d-d max (nm),  (M -1 cm -1 )	 CD  (M -1 cm -1 ) max (nm),
				305, -0.64
	CuH 3 L 2	25.64(1)	640, 71.6	545, 0.27 635, -0.17
				305, -1.19
	CuH 2 L 2	22.50(2)	625, 91.6	545, 0.59 630, -0.31
				310, -0.89
	CuHL 2	18.97(2)	625, 88.5	545, 0.73 635, -0.22
				310, -0.85
	CuL 2	12.77(3)	625, 90.3	550, 0.74 635, -0.20
				310, -0.69
	CuH -1 L 2	5.40(3)	627, 91.9	550, 0.66 640, -0.18
	CuH -2 L 2 -5.21(4)	650, 109.5	630, -1.53 780, 0.82
	Cu 2 L 2	17.84(1)	650, 118.4	310, -0.80 550, 0.66
				315, -0.61
	Cu 2 H -1 L 2 12.56(1)	665, 142.3	560, 0.90 705, 0.47
				305, -3.09
	Cu 2 H -2 L 2 5.91(1)	605, 179.5	525, 1.01 665, -0.59
				310, -2.34
	Cu 2 H -3 L 2 -3.42(1)	630, 180.8	570, 1.01 650, 0.96

Table 4 . 3

 43

	g o	A o /G	a N	g x	g y	g z	A x /G A y /G A z /G a x N	a y N	a z N
	(a) 2.1121	71.5	11.6	2.0448	2.0526	2.2285	17.4	8.6	178.9	12.0	10.0	10.0
			11.6 11.6							12.0	10.0	10.0
										13.1	11.3	9.3
	(b) 2.105	w=165 G		2.106			w=180G
	(c) 2.1117	61.8	13.9	2.097(1) 2.012(1) 2.225(1)	34.2(2) 20.0(4) 140.9(2)
			13.9
			13.9

. Calculated EPR parameters of component spectra detected in the Cu(II)-L 2 system. The component spectra (a), (b) and (c) are related to the complexes CuH x L 2 (x = 2, 1, 0, -1), Cu 2 H x L

2

(x = -1, -2, -3) and CuH -2 L 2 , respectively.

  2 ]  = [C 24 H 27 N 10 O 9 Cu]  and [Cu 2 H -2 L 2 ]  = [C 24 H 25 N 10 O 9 Cu 2 ]  . However, differences between the estimated (662.13, 723.04) and measured (663.26, 725.21) molecular weights were observed, which were attributed to the reduction of Cu 2+ to Cu + and the corresponding uptake of a proton during the ionization process (see Section 4.1.2). : calculated spectra of the corresponding complexes ([C 24 H 27 N 10 O 9 Cu]  and [C 24 H 25 N 10 O 9 Cu 2 ]  ).

	A			662.13		B	725.21	723.04
		663.26
								722	724	726	728 m/z M/z	730	732
			660 662 664 666 668 670
				M/z m/z
	660	662	664 M/z 666 m/z	668	670	723	725	727 M/z m/z	729		731

Figure 4.24. MALDI-TOF MS spectra measured in the Cu(II)-L 2 1:1 (A) and 2:1 (B) systems. Insert

Table 4 . 4 .

 44 Formation and deprotonation constants of zinc(II)-L 2 complexes (estimated errors in parentheses (last digit), I = 0.1 M NaCl, T = 298 K).

2 

 2 ZnH -1 L 2 + H + .

		log	pK
	ZnHL 2	12.26 (8)	6.50
	ZnL 2	5.76(6)	8.74
	ZnH -1 L 2	-2.98(8)	10.26
	ZnH -2 L 2

Table 4 . 5 .

 45 Protonation constants (log) and pK values of the ligands nta1his and nta1his-NH 2 (I = 0.1 M NaCl, T = 298 K, estimated error of last digits in parentheses).

		nta1his		nta1his-NH 2
		log	pK	log	pK
	H 3 L	16.24(2)	2.30	14.80(2)	2.15
	H 2 L	13.94(1)	6.44	12.65(1)	5.66
	HL	7.50(1)	7.50	6.99(1)	6.99

Table 4 . 7 .

 47 Protonation constants and pK values of tren1his and tren2his (I = 0.1 M NaCl, T = 298 K, estimated error of last digits in parentheses).

		tren1his	tren2his
		log	pK	log	pK
	H 6 L			36.46(1)	2.33
	H 5 L			34.13(1)	4.62
	H 4 L	31.04(1) 5.03 29.51(1)	5.41
	H 3 L	26.01(1) 7.07 24.10(1)	6.82
	H 2 L	18.94(1) 9.04 17.28(1)	7.69
	HL	9.90(1) 9.90	9.59(1)	9.59

1

H NMR titration of ligand L

3

(in H 2 O,

[L 3

] tot = 0.0018 M, I = 0.1 M NaCl, T = 298 K).

03 pH 6.62 pH 5.84 pH 5.25 pH 4.79 pH 4.52 pH 3.96 pH 3.29 pH 2.57 pH 9.03 pH 9.50 pH 10.14 pH 10.37

			CH 2 -NHCO	N tert -CH 2 free CH 2 -NH 2
	C 2 H	C 5 H	CH	CH 2	N tert -CH 2 funct

Table 4 . 8 .

 48 Formation constants, deprotonation constants (I = 0.1 M NaCl, T = 298 K, estimated error of last digits in parentheses) and spectroscopic (UV-Vis, CD and EPR) parameters of the main copper(II)-L 3 complexes.

		log	pK	 max d-d (nm),  (M -1 cm -1 )	 max CD (nm),  (M -1 cm -1 )	g o , calc *
	CuH 3 L 3	30.735(2)	3.197
	CuH 2 L 3	27.538(1)	4.984	640, 53.1	305, -0.302 635, 0.212
	CuHL 3	22.554(4)	7.173	635, 75.6	315, -0.616 675, 0.573	2.096
	CuL 3	15.381(6)	7.574	580, 88.0	675, 0.380
	CuH -1 L 3	7.807(5)	11.077	555, 156.3	315, 0.473 595, 0.223	2.097
	CuH -2 L 3	-3.27(1)
	Cu 2 L 3	21.906(4)	4.741	580, 119.4	300, -0.884 750, 0.263
	Cu 2 H -1 L 3	17.165(3)	8.419	570, 161.1	300, 1.421 700, 0.246	2.106
					310, 0.735
	Cu 2 H -2 L 3	8.746(4)	9.368	575, 168.4	515, -0.180
					670, 0.347
	Cu 2 H -3 L 3	-0.622(5)		585, 205.1	300, -0.698 665, 0.307	2.108
	Cu 3 H -1 L 3 2 Cu 3 H -2 L 3 2	36.72(2) 29.34(1)	7.38
	Cu 2 H -1 L 3 -CuHL 3			546, 11.3
	*g o,calc = (g x +g y +g z )/3, anisotropic parameters and fitted spectra can be found in
	Appendix A.23. and Table A.1.

  : calculated spectra of the corresponding complexes: [C 18 H 31 N 10 O 2 Cu 2 ] + (A), [C 18 H 29 N 10 O 2 Cu 2 ] + (B) and [C 36 H 59 N 20 O 4 Cu 3 ]

	[Cu I	3 H -2 L 3	2 ] +	1029.49			1024.29		1026.29	[Cu II	3 H -5 L 3	2 ] +
		1027.48				1022	1026	m/z	1030	1034
	1026	1028	1030		1032	1034	1036
				m/z
			545.21			543.11			[Cu II	2 H -3 L 3 ] +
	[Cu I	2 H -1 L 3 ] +
					542	544		546	548	550
									m/z
	542 544 546 548 550 552 554 556
					m/z
		483.25		482.20	[Cu II H -1 L 3 ] +
	[Cu I L 3 ] +		480	482	484	486	488
							m/z
		482	484	486		488		490
					m/z

A B C Figure 4.35. MALDI-TOF MS spectra measured at Cu(II)-L 3 1:1 (A), 2:1 (B) and 3:2 (C) ratios.

Insert+ (C).

Table 4 . 10 .

 410 Formation constants, deprotonation constants (I = 0.1 M NaCl, T = 298 K, estimated error of last digits in parentheses) and spectroscopic (UV-Vis, CD and EPR) parameters of the copper(II)-L 4 complexes.

		log	pK	 max d-d (nm),  (M -1 cm -1 )	 max CD (nm),  (M -1 cm -1 )	g o , calc *
	CuH 2 L 4 27.181(6) 3.951	~670, 60.6	~720, 0.12
	CuHL 4	23.230(8) 5.931	585, 118.6	535, 0.167 645, -0.188	2.113
					545, 0.076
	CuL 4	17.299(9) 6.529	615, 142.9	605, -0.016
					695, 0.118
	CuH -1 L 4	10.77(1)		632, 169.1	300, -1.02 635, 0.259	2.112

*g o,calc = (g x +g y +g z )/3, anisotropic parameters and fitted spectra can be found in Appendix A.23. and Table A.1.

Table 4 .

 4 11. Formation and deprotonation constants of Zn(II)-L 4 complexes (I = 0.1 M NaCl, T = 298 K, estimated error of last digits in parentheses).

	log	pK
	ZnH 2 L 4 23.45(3) 6.45
	ZnHL 4 17.00(3)
	ZnH -1 L 4 4.16(2) 10.73
	ZnH -2 L 4 -6.57(4)

Table 4 .

 4 12. Experimental conditions used in the kinetic measurements of H 2 DTBC oxidation by Cu(II)-L systems (in H 2 O/EtOH 50/50%, I = 0.1 M NaCl-buffer (MES/HEPES/CHES), T = 298 K in all experiments).

	system	[S] o (M) [complex] tot (M) pH corr	varying condition
	Cu(II)-L 1 3:2	1.5×10 -3	5.0×10 -5		pH corr
			5.0×10 -5	7.8	[S] o
		5.0×10 -3		7.8	[complex] tot
		5.0×10 -4	4.8×10 -5	7.8	[O 2 ]
	Cu(II)-L 2 2:1	7.0×10 -4	5.0×10 -5		pH corr
			1.0×10 -5	8.9	[S] o
		4.0×10 -4		8.9	[complex] tot
		8.0×10 -4	5.0×10 -5	8.9	[O 2 ]
	Cu(II)-L 3 1:1	8.0×10 -4	5.0×10 -5		pH corr
			1.0×10 -5	7.4	[S] o
	Cu(II)-L 3 2:1	4.0×10 -4	1.0×10 -5		pH corr
			5.3×10 -6	8.7	[S] o
		5.4×10 -4		8.7	[complex] tot
		5.5×10 -4	1.0×10 -5	8.7	[O 2 ]

Table 4 .

 4 13.Comparison of catecholase activity of some previously investigated copper(II)-ligand systems along with the catechol oxidase enzyme.

	Cu(II)-ligand systems (pH)	k cat (s -1 )	K M (M)	k cat /K M (M -1 s -1 )	solvent, T ( • C)	ref.
	Cu(II)-Ac-HHGH 1:1 (10.18)	0.063	not rep.	not rep.	MeOH/H 2 O 86/14, 25	[65]
	Cu(II)-MDVFMKGLSKAKEGV-NH 2 1:2 (7.4)	0.009	1.8×10 -3	5.0	MeOH/H 2 O 80/20, 25	[80]
	Cu(II)-B1 a 2:1 (8.50)	0.051	9.44×10 -3	5.47	MeOH/H 2 O 97/13, 25 [136]
	Cu(II)-B2 a 2:1 (8.50)	0.0078	9.5×10 -4	8.1	MeOH/H 2 O 97/13, 25 [136]
	Cu(II)-B3 b 1:2	0.00596	2.6×10 -4	23.37	DMF, 20	[64]
	Cu(II)-B4 c 2:1	0.25	6.4×10 -3	39.1	MeCN, 25	[137]
	Cu(II)-L 3 1:1 (7.4)	0.0056	7.09×10 -5	79.4	EtOH/H 2 O 50/50, 25
	Cu(II)-B5 d 2:4:2	0.0426	1.45×10 -4	293.8	MeOH, 25	[138]
	Cu(II)-B6 e 2:2	2.63	5.90×10 -3	445.8	MeOH, not rep.	[69]
	Cu(II)-L 1 3:2 (7.8)	0.071	1.12×10 -4	632.1	EtOH/H 2 O 50/50, 25
	Cu(II)-B7 f 2:1	1.916	1.2×10 -3	1597	MeCN, 25	[68]
	Cu(II)-B8 g 2:1 (8.0)	1.806	7.5×10 -4	2408	MeOH/H 2 O 29/1, 20	[67]
	Cu(II)-L 2 2:1 (8.9)	0.387	1.07×10 -4	3625	EtOH/H 2 O 50/50, 25
	Cu(II)-B9 h 2:1	9	2.3×10 -3	3913	MeOH, 20	[71]
	Cu(II)-L 3 2:1 (8.7)	0.157	3.61×10 -5	4362	EtOH/H 2 O 50/50, 25
	Cu(II)-B10 i 2:1	8	7×10 -4	11429	MeOH,

Table 4 .

 4 14. UV-Vis spectroscopic parameters and deprotonation constants of 4-nitrocatechol (H 2 4NC) substrate analog (EtOH/H 2 O 50/50, T = 298 K, I = 0.1 M NaCl). Calculated individual molar spectra in

  Proposed mechanism of catalytic oxidation of 3,5-di-tert-butylcatechol by L1 and L 2 ligand-copper(II) systems.

		L 2
		Cu 2+	Cu 2+
			H 2 DTBC
			-2H +
		H 2 DTBC	DTBQ
			H 2 O 2
	L 2		L 2
	Cu 2+ (O 2 2-)	Cu 2+	Cu +	Cu +
		O 2
	Scheme 4.13.

Table 4 .

 4 15. Michaelis-Menten parameters (estimated errors in parentheses) of H 2 DTBC oxidation catalyzed by the investigated copper(II)-peptide systems, along with the composition and coordination mode of the suggested active species.

	peptide (pH)	major species and its coordination mode	k cat / s -1	K M / mM k cat /K M / M -1 s -1
	P3, pH 8.9	CuL {N im ,2N  }	0.34(2)	0.90(1)	378
	P5, pH 9.2	CuH 2 L {2N im ,2N  }	0.043(3)	0.25(5)	172
	P2, pH 8.3	CuL {2N im ,2N  }	0.18(1)	1.6(3)	113
	P1, pH 8.3	CuL {2N im ,2N  }	0.27(1)	3.7(4)	73
	P4, pH 8.2	CuL {2N im ,2N  }	0.044(1)	0.8(1)	55

Table 4 .

 4 16. IC 50 and k values of Cu(II)-tripodal peptide systems, together with those of other copper(II)-linear peptide complexes and the native Cu,Zn-SOD enzyme. k calculated as k NBT ×[NBT]/IC 50 (k NBT = 3×10 4 M -1 s -1 , see Section 3.3.2.)

	complex	IC	50 (M)	k (M -1	s -1	) a	Ref.
	Cu,Zn-SOD (pH 6.8)	0.0045	3.3×10 8	[65]
	Cu(II)-L 3 2:1 (pH 7.4)		0.13	4.6×10 7
	Cu(II)-P5 1:1 (pH 7.4)		0.10	3.0×10 7
	Cu(II)-L 3 1:1 (pH 7.4)		0.20	3.0×10 7
	Cu(II)-L 2 2:1 (pH 7.4)		0.13	2.3×10 7
	Cu(II)-L 1 1:1 (pH 7.4)		0.17	1.8×10 7
	Cu(II)-P4 1:1 (pH 7.0)		0.11	1.4×10 7	[88]
	Cu(II)-P2 1:1 (pH 7.2)		0.50	1.2×10 7
	Cu(II)-P1 (pH 7.8)		0.65	9.2×10 6
	Cu(HPO 4 ) (pH 7.4)		1.06	6.2×10 6	[153]

a

  ) Nous avons également voulu trouver des liens entre les activités catalytiques et les structures des complexes métalliques. Dans ce but, il était important de comparer nos systèmes aux complexes catalytiques actifs formés avec des peptides linéaires. Ces résultats peuvent fournir des informations importantes pour le développement de nouveaux mimes enzymatiques plus efficaces.

Cu(II)-L 1 3:2 (7.8) 0.071 1.12×10 -4 632.1 EtOH/H 2 O 50/50, 25

  3 2:1, pH corr 8.7). Les lignes continues correspondent aux courbes calculées.Tableau IV.1. L'activité catécholase de quelques systèmes cuivre(II)-L précédemment étudiés ainsi que celle de l'enzyme native.

	Cu(II)-ligand systems (pH)	k cat (s -1 )	K M (M)	k cat /K M (M -1 s -1 )	Solvent, T ( • C)	Ref.
	Cu(II)-Ac-HHGH 1:1 (10.18)	0.063	not rep.	not rep.	MeOH/H 2 O 86/14, 25	9
	Cu(II)-MDVFMKGLSKAKEGV-NH 2 1:2 (7.4)	0.009	1.8×10 -3	5.0	MeOH/H 2 O 80/20, 25	37
	Cu(II)-B1 a 2:1 (8.50)	0.051	9.44×10 -3	5.47	MeOH/H 2 O 97/13, 25	91
	Cu(II)-B2 a 2:1 (8.50)	0.0078	9.5×10 -4	8.1	MeOH/H 2 O 97/13, 25	91
	Cu(II)-B3 b 1:2	0.00596	2.6×10 -4	23.37	DMF, 20	76
	Cu(II)-B4 c 2:1	0.25	6.4×10 -3	39.1	MeCN, 25	90
	Cu(II)-L 3	1:1 (7.4)	0.0056	7.09×10 -5	79.4	EtOH/H	2 O 50/50, 25
	Cu(II)-B5 d 2:4:2	0.0426	1.45×10 -4	293.8	MeOH, 25	89
	Cu(II)-B6 e 2:2	2.63	5.90×10 -3	445.8	MeOH, not rep.	88
	Cu(II)-B7 f 2:1	1.916	1.2×10 -3	1597	MeCN, 25	87
	Cu(II)-B8 g 2:1 (8.0)	1.806	7.5×10 -4	2408	MeOH/H 2 O 29/1, 20	71
	Cu(II)-L 2	2:1 (8.9)	0.387	1.07×10 -4	3625	EtOH/H	2 O 50/50, 25
	Cu(II)-B9 h 2:1	9	2.3×10 -3	3913	MeOH, 20	68
	Cu(II)-L 3	2:1 (8.7)	0.157	3.61×10 -5	4362	EtOH/H	2 O 50/50, 25
	ibCO i (6.5)	2293	2.5×10 -3	917200	H 2 O, 20	79

Cu 2+ Scheme 4.11. Proposed structures of the main copper(II)-L 4 complexes.

Acknowledgements

138

ACKNOWLEDGEMENTS

I am grateful to the head of Department of Inorganic and Analytical Chemistry, Dr. Gábor Galbács, as well as the leader of the Bioinorganic Research Group, Prof. Tamás Kiss, for allowing me to carry out my PhD work in their laboratories at the University of Szeged. Equally, I thank Prof. Philippe Gros for accepting me as doctoral student in his group of Laboratoire Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC UMR 7565), in the University of Lorraine. I wish to acknowledge the financial support provided by the French Government and Campus France, enabling my co-tutelle PhD program. I wish to express my gratitude to my main supervisor, Prof. Tamás Gajda, who assisted in my scientific development since my second undergraduate year. His advice and scientific knowledge accompanied and helped me along my doctoral years. He spent considerable amount of his time on the interpretation and discussion of my results, would that be either for presentations, papers or my BSc/Master/PhD thesis. I am thankful to Prof. Bernard Henry and Dr. Florence Dumarçay, my supervisors from the University of Lorraine, for accepting the direction of my PhD thesis, as well as for their advice, corrections and opinions.

My deepest gratitude goes to my co-supervisor, Dr. Katalin Selmeczi. Her joining in the direction of my work was a real inspiration; I am thankful for her valuable help around my experiments, as well as for the scientific discussions and the corrections of papers and manuscripts. Her assistance and support was imperative in the months of my stay in Nancy, not to mention her huge contribution to my improvement regarding French language.

Many thanks to all our scientific partners in the University of Szeged, the Biological Research Centre in Szeged (Dr. Zsuzsanna Darula, Dr. Tibor Páli), the University of Debrecen (Dr. István Bányai) and the University of Lorraine (Dr. André Merlin, Dr. Gabriel Krier, Lionel Vernex-Loset, Sophie Poinsignon, Fabien Lachaud, Pascal Lemière). I am especially thankful to Dr. Nóra May from the Hungarian Academy of Science, for her help in the EPR measurements and evaluations, and also to Stéphane Parant, for his technical help in the laboratory concerning my experiments.

I owe thanks to my family, highlighting my mother, and my partner, Balázs. Their continuous support gave me strength even in the hardest moments. I wish to thank the invigorating presence of my friends, especially: Gergely Samu, Attila Kormányos, Nóra Grecsó, Gabriella Fiser and Fernanda Haffner. Every single coffee/tea/pizza we've had together contributed to this work! Finally, thank you all, fellows in the PhD room, for the laughs and chats: Orsi, Ria, Edit, Dani, Levi, Feri. You of all people know the best the joys and hardship of this journey.

TFA -trifluoroacetic acid TIS -triisopropylsilane trentris(2-aminoethyl)amine Trt -trityl

Methods

CD -Circular Dichroism EPR -Electron Paramagnetic Resonance ESI-MS

Copper(II) and zinc(II) complexes of L 1 and L 2 symmetrical tripodal ligands have been tested in hydrolytic functions, using the activated phosphoesters BNPP, NPP, HPNP and DNPEP. The experiments were carried out in aqueous, buffered solutions at T = 25 C, adjusted to the pH corresponding to major metal-ligand species (see Table A.2). The formation of the colored products was followed spectrophotometrically at 400 nm. Observed pseudo-first order and pseudo-second order reaction rate constants (k obs,corr , k') were calculated according to the initial reaction rates method, and given in Table 4.17. All values have been corrected with the rate constant of autohydrolysis (k obs,corr = k obs -k auto ).

Considering the obtained rate constants, and taking into account the magnitude of catalytic activity of efficient phosphoesterase mimics from the literature [92,94,154], the investigated metal-ligand systems demonstrate low-medium hydrolytic activities. Generally, it can be stated that copper(II) complexes exhibit somewhat higher hydrolytic activities; species formed at higher pH perform better than those in the acidic/neutral pH range. The relatively weak activities are most likely related to the high number of donor groups of the tripodal ligands, and thus the saturated coordination sphere around the metal ion, preventing the formation of the nucleophilic OH -group and the coordination/activation of the substrate.

Since none of the studied systems presented significantly high or selective hydrolytic properties, further detailed kinetic investigation was not pursued. Cu(II)-L

1.18×10 -5 1.26×10 -7 3.86×10 -4 1.23×10 -7 3.77×10 -4 2.53×10 -7 8.52×10 -4 Zn(II)-L 1 1:1, pH 8.2 ZnH -1 L 1 {2NH 2 ,2N im ,OH -} --1.95×10 -7 6.00×10 -4 1.74×10 -7 5.34×10 -4 6.37×10 -7 2.15×10 -3 Zn(II)-L

Abstracts

One of the most important directions of modern bioinorganic research is the development of artificial enzymes. One pathway of enzyme modeling strategy is the study of amino acid sequences present in the active centers of metalloenzymes. Linear peptides, however, have their limitations in reconstituting the active centers of metalloenzymes, since they do not possess the well-defined three dimensional structure, therefore their structure is vulnerable towards amide nitrogen coordination/hydrolysis. Improvement of metal binding capabilities of linear peptides can be obtained by e.g. their functionalization with tripodal ligands. Tripodal compounds may provide a rigid, less flexible platform for the coordinating amino acid side chains, creating pre-organized metal binding sites.

In my thesis, I present synthesis and characterization of histidine containing tripodal peptide ligands and their complex formation in presence of copper(II) and zinc(II). Solution equilibrium was studied with pH potentiometric measurements, and several spectroscopic methods were used for structural characterization (UV-Vis, CD, ESR, NMR and MS methods). Beside thermodynamic and structural characterization, enzyme mimicking catalytical properties of the complexes have also been investigated (catechol oxidation, superoxide dismutation).

Our results demonstrated that tripodal peptide ligands are capable of enhancing the stability of metal-peptide complexes, and they may provide convenient structures to efficiently mimic the catalytic functions of enzymes. With thorough and systematical solution equilibrium and spectroscopic studies, we uncovered the driving forces of metal coordination, and established the impact of the tripodal structure in stability, structure and catalytic properties of the forming complexes. Our findings confirm the beneficial effect of tripodal scaffolds in peptide-type ligandmetal complexes, and emphasize the possibilities lying within tripodal peptides in the field of enzyme mimicking.

Keywords: tripodal ligands, peptide complexes, copper(II), zinc(II), enzyme mimicking De nos jours, un des objectifs importants de la recherche bioinorganique moderne est le développement d'enzymes artificielles. L'étude séquentielle des acides aminés présents dans le centre actif des métalloenzymes peut présenter une voie possible de la stratégie de modélisation enzymatique. Cependant, les peptides linéaires ont leurs limites lors de la reconstitution des centres actifs des métalloenzymes : ils ne possèdent pas la structure tridimensionnelle bien définie, par conséquent leur structure est vulnérable vis-à-vis de la coordination ou de l'hydrolyse des azotes amidiques. La capacité de coordination des métaux par des peptides linéaires peut être améliorée, par exemple, en les attachant à une plateforme tripodale. Les composés tripodaux peuvent assurer une organisation structurale rigide ou moins flexible pour des chaînes latérales des acides aminés, créant ainsi des sites de coordination pré-organisés pour les métaux.

Dans cette thèse, la synthèse et la caractérisation des ligands peptidiques tripodaux contenant de l'histidine et la formation des complexes en présence de cuivre(II) et de zinc(II) sont présentées. Les propriétés acido-basiques ont été étudiées par potentiométrie et différentes techniques spectroscopiques ont été utilisées pour la caractérisation structurale (UV-Vis, CD, ESR, RMN et MS). Outre que la caractérisation thermodynamique et structurale, des propriétés catalytiques des complexes en réaction enzymatiques (oxydation du catéchol, dismutation du superoxyde) ont également été étudiées.

Nos résultats ont démontré que les ligands peptidiques tripodaux sont capables d'améliorer la stabilité des complexes métalliques et qu'ils peuvent fournir des structures adéquates pour mimer efficacement les fonctions catalytiques des enzymes. Grâce aux études approfondies et systématiques des propriétés acido-basiques et spectroscopiques, nous avons mis en évidence les forces motrices de la coordination des métaux et établi l'impact de la structure tripodale sur la stabilité, la structure et les propriétés catalytiques des complexes formés. Nos résultats confirment l'effet bénéfique des plateformes tripodales durant la complexation des métaux, et soulignent les possibilités qui s'offrent aux peptides tripodaux dans le domaine de la biomimétisme.

Mots-clés: ligands tripodaux, complexes peptidiques, cuivre(II), zinc(II), mime enzymatique