Design and Evaluation of Seven-coordinate Manganese and ... · Design and Evaluation of...

I

Design and Evaluation of Seven-coordinate

Manganese and Iron Complexes, and Fullerene

derivatives, as SOD mimetics

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades

vorgelegt von

Gao-Feng Liu

aus

VR China

Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics

II

Als Dissertation genehmigt von den Naturwissenschftlichen Fakultäten der Friedrich

-Alexander-Universität Erlangen-Nürnberg.

Tag der mündlichen Prüfung: 02.05.2008

Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch

Erstberichterstatter: Prof. Dr. Dr. h. c. R. van Eldik

Zweitberichterstatter: Prof. Dr. Lutz Dahlenburg

III

Acknowledgements

This study was carried out from February 2003 to February 2008 at the

Institute of Inorganic Chemistry at the Friedrich-Alexander-University of

Erlangen-Nürnberg under the supervision of Prof. Dr. Dr. h. c. mult Rudi van

Eldik.

I would like to express my sincere gratitude to my supervisor, Dr. Ivana

Ivanović-Burmazović for her essential guidance, her never-ending enthusiasm, and

permanent encouragement throughout my study. At the same time I am thankful to

Prof. Dr. Dr. h. c. mult Rudi van Eldik for his kind and helpful discussion.

Warm thanks are given to the whole group for the friendly working

atmosphere.

Thanks to the Friedrich-Alexander-University of Erlangen-Nürnberg and the

DFG within SFB 583 “Redox-active Metal Complexes” for the financial support.

Finally I am grateful to my parents and to my wife for endless support which

has been valuable during my study.

Gao-Feng Liu


IV

Publications

1 Gao-Feng Liu, Ralph Puchta, Frank W. Heinemann, Ivana Ivanović-Burmazović,

Ligand Electronic Properties in the Control of Redox Behavior and Reactivity

toward Superoxide in Seven-Coordinate Manganese Complexes, Chemical

Communication (Submitted)

2 Gao-Feng Liu, Miloš Filipović, Ivana Ivanović-Burmazović, Florian Beuerle, Patrick

Witte, Andreas Hirsch, Highly Catalytic Metal-Free Superoxide Dismutation mimics

from Dendritic Monoadducts of C60, Angewandte Chemie (in print) 3 Gao-Feng Liu, Miloš Filipović, Frank W. Heinemann and Ivana Ivanović-Burmazović

Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate

Chelates and their Superoxide Dismutase Activity, Inorganic Chemistry, 2007, 46,

8825-8835. 4 David Sarauli, Roland Meier, Gao-Feng Liu, Ivana Ivanovic-Burmazovic, Rudi van

Eldik, Effect of Pressure on Proton-Coupled Electron Transfer Reactions of

Seven-Coordinate Iron Complexes in Aqueous Solutions, Inorganic Chemistry, 2005,

44, 7624-7633.

Conference contributions

1 Poster “Sub-millisecond Mixing Stopped-Flow Configuration to SOD Activity”,

Inorganic Reaction Mechanism Meeting, organised by the Royal Society of

Chemistry, Athens, Greece, January 2004.

2 Poster “Cryo-stopped-flow measurements for rapid inorganic reactions”,

Inorganic Reaction Mechanism Meeting, organised by the Royal Society of

Chemistry, Athens, Greece, January, 2004.

V

3 Oral Presentation “Seven-coordinate Iron and Manganese Complexes and

Reactivity towards Superoxide”, Conference on Coordination Chemistry of

China, organised by the Chinese Society of Chemistry, Guangzhou, China,

October, 2005.

4 Poster “Superoxide Dismutase Mimetics. From Seven-coordinate Iron and

Manganese Complexes to Fullerenes”, Inorganic Reaction Mechanism Meeting,

organised by the Royal Society of Chemistry, Krakow, Poland, January, 2006.

5 Poster “Design and Evaluation of Seven-coordinate Iron and Manganese

Complexes, Fullerenes as SOD mimics”, SFB-Symposium on Redox-Active

Metal complexes: Control of Reactivity via Molecular Architecture, Erlangen,

Germany, March, 2007.

Abbreviations

SODs superoxide dismutases

ROS reactive oxygen species

RNS reactive nitrogen species

SAR Structure-Activity-Relationship

His L-Histidine

Asp L-Aspartic acid

Me2[15]pyridinaneN5 trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo

[12.3.1]-octadeca-1(18),14,16-triene)

H2dapsox 2,6-diacetylpyridine-bis(semioxamazide)

H2Dcphp Pyridine-2,6-biscarboxylic acid-bis((N′-2-pyridine-2-yl)

hydrazide)

Daphp 2,6-diacetylpyridine-bis(2-pyridylhydrazone)

Hdapmp [1-(6-acetyl-2-pyridinyl) ethylidene] hydrazone


VI

salen N,N´-ethylenebis(salicylideniminate)

TBAP tetrakis(4-benzoic acid)porphyrin

Tempol 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl

t1/2 half life

λ wavelength (nm)

δ chemical shift(NMR)

ν stretching mode(IR)

A absorbance

PBP pentagonal-bipyramidal

DD distorted-dodecahedron

IC50 half maximal inhibitory concentration

K equilibrium constant

kcat catalytic rate constant

kobs observed rate constant

OPs oxidation potentials

NHE normal hydrogen electrode

SHE standard hydrogen electrode

CV cyclic voltammetry

NBT nitro blue tetrazolium

UV ultraviolet

DFT density functional theory

IR infrared spectroscopy

NMR nuclear magnetic resonance

MS mass spectrometry

VII

Contents ACKNOWLEDGEMENTS......................................................................................................................................III PUBLICATIONS .................................................................................................................................................. IV CONFERENCE CONTRIBUTIONS ........................................................................................................................ IV ABBREVIATIONS .................................................................................................................................................V

CHAPTER 1 ...........................................................................................................................................................1

INTRODUCTION..................................................................................................................................................1

1.1 SUPEROXIDE AND SUPEROXIDE DISMUTASES (SODS) ...........................................................................1 1.1.1 Superoxide and its toxic effects to damage the cell ............................................................................1 1.1.2 Superoxide Dismutases (SODs) and their Active Metal Center Structures ..........................................3

1.2 DEVELOPMENT AND CONSIDERATIONS OF SYNZYMES AS SOD MIMICS ..............................................6 1.2.1. Manganese(III) Metalloporphyrins ..................................................................................................8 1.2.2. Manganese(III) salen Complexes .....................................................................................................9 1.2.3. Nitroxide.........................................................................................................................................10 1.2.4. Manganese(II) (pentaazamacrocyclic ligand)-Based Complexes ..................................................11

1.3 CHARACTERIZATION ARRAYS OF SOD ACTIVITY ...............................................................................12 1.3.1 Indirect Assay .................................................................................................................................12 1.3.2 Direct Arrays..................................................................................................................................12 1.3.3 Cyclic Voltammetry Method ...........................................................................................................13

1.4 FULLERENE AND ITS ANTIOXIDANT ABILITY ........................................................................................14 1.5 AIMS OF THIS THESIS .............................................................................................................................18 1.6 REFERENCES ..........................................................................................................................................19

CHAPTER 2 .........................................................................................................................................................25

SEVEN-COORDINATE IRON AND MANGANESE COMPLEXES WITH ACYCLIC AND RIGID PENTADENTATE CHELATES AND THEIR SUPEROXIDE DISMUTASE ACTIVITY.........................25

2.1 ABSTRACT ..............................................................................................................................................25 2.2 INTRODUCTION ......................................................................................................................................26 2.3 EXPERIMENTAL SECTION......................................................................................................................29 2.4 RESULTS AND DISCUSSION ....................................................................................................................35

2.4.1 Stability of Superoxide Anion in H2O-DMSO.................................................................................35 2.4.2 Structures .......................................................................................................................................37 2.4.3 Electrochemistry.............................................................................................................................45 2.4.4 Reaction with superoxide in DMSO ...............................................................................................48 2.4.5 Reaction with superoxide in aqueous solution ...............................................................................51

2.5 CONCLUSIONS ........................................................................................................................................53 2.6 REFERNCES ............................................................................................................................................54

CHAPTER 3 .........................................................................................................................................................60

LIGAND ELECTRONIC PROPERTIES IN THE CONTROL OF REDOX BEHAVIOR AND REACTIVITY TOWARD SUPEROXIDE IN SEVEN COORDINATE MANGANESE COMPLEX........60

3.1 ABSTRACT ..............................................................................................................................................60


VIII

3.2 INTRODUCTION ......................................................................................................................................61 3.3 EXPERIMENTAL SECTION......................................................................................................................62 3.4 RESULTS AND DISCUSSION ....................................................................................................................66

3.4.1 Structure .........................................................................................................................................66 3.4.2 Electrochemistry.............................................................................................................................70 3.4.3 Reactions with Superoxide in DMSO .............................................................................................72 3.4.4 Modelling via DFT Calculations:...................................................................................................74

3.5 CONCLUSIONS ........................................................................................................................................76 3.6 REFERENCES ..........................................................................................................................................76

CHAPTER 4 .........................................................................................................................................................79

STRUCTURAL FEATURES IN CONTROL OF REACTIVITY TOWARD SUPEROXIDE IN MANGANESE AND IRON COMPLEXES.......................................................................................................79

4.1 ABSTRACT ..............................................................................................................................................79 4.2 INTRODUCTION ......................................................................................................................................80 4.3 EXPERIMENTAL SECTION......................................................................................................................81 4.4 RESULTS AND DISCUSSION ....................................................................................................................85

4.4.1 Studies on the complex 7 ................................................................................................................85 4.4.2 Studies on the complex 8 ................................................................................................................91

4.5 CONCLUSION..........................................................................................................................................96 4.6 REFERENCES ..........................................................................................................................................96

CHAPTER 5 .........................................................................................................................................................99

HIGH CATALYTIC ACTIVITY OF DENDRITIC C60 MONOADDUCTS IN METAL-FREE SUPEROXIDE DISMUTATION........................................................................................................................99

5.1 ABSTRACT ..............................................................................................................................................99 5.2 INTRODUCTION ....................................................................................................................................100 5.3 EXPERIMENTAL SECTION....................................................................................................................101 5.4 RESULTS AND DISCUSSION ..................................................................................................................107 5.5 CONCLUSION........................................................................................................................................115 5.6 NOTE AND REFERENCES......................................................................................................................116

SUMMARY ........................................................................................................................................................118

ZUSAMMENFASSUNG ...................................................................................................................................122

Chapter 1 Introduction

1

Chapter 1

Introduction

1.1 Superoxide and Superoxide Dismutases (SODs)

1.1.1 Superoxide and its toxic effects to damage the cell

Oxygen is vital to life, but as a diatomic molecule it is remarkably unreactive.

Normally when the terminal oxidases (cytochrome c oxidase) react with oxygen, four

electrons are transferred and water is formed. Occasionally oxygen can also react with

other electron transport components, herein only one electron is transferred and it

causes the overproduction of the superoxide anion in our bodies. It is a reducing agent

in the anionic form [Eq. (1)], and an oxidant in the protonated form (pKa (HO2) = 4.69)

[Eq. (2)]

O2• + e- + H+ HO2 E° = + 0.9 V (2)– –

O2• O2 + e- E° = - 0.16 V (1)–

O2• + e- + H+ HO2 E° = + 0.9 V (2)O2• + e- + H+ HO2 E° = + 0.9 V (2)– –

O2• O2 + e- E° = - 0.16 V (1)–O2• O2 + e- E° = - 0.16 V (1)O2• O2 + e- E° = - 0.16 V (1)–

According to the stoichiometry of O2

·– and H2O2 dismutation reactions, in plant

tissues about 0.9 to 1.5% of the total oxygen uptake proceeds through the formation of

the free radical intermediates of the partial reduction of oxygen.[1] Although some of

the O2·– generated by neutrophiles and other immune cells is used to kill pathogenic

bacteria or parasites, under the condition of bad lifestyle (smoking, over exercise, over

dieting, unbalanced diet, inadequate rest, irregular sleeping patterns and stress) or other


2

external factors (UV rays, radiation, environmental pollution), O2·– will be

overproduced. Although normal forms of life maintain a reducing environment in their

cells, disturbances in this normal redox state can cause toxic effects to damage all

components of the cell. The tissue toxicity from extracellular superoxide generation

seems to be based on three aspects: i) its direct reactivity with numerous types of

biological molecules (lipid, DNA, RNA, catecholamines, steroids, etc.); ii) its

dismutation to form H2O2, in which H-atoms were absorbed from such key biological

targets as catecholamines or the allylic CH in lipid; iii) its capacity to inactivate

iron-sulfur cluster containing enzymes (which are critical in a wide variety of

metabolic pathways), thereby liberating free iron in the cell, which can in

iron-catalyzed Fenton reaction between trace amounts of ferrous ions and H2O2

produce the highly reactive hydroxyl radical.

Moreover, superoxide also is the substrate for the generation of a variety of

reactive oxygen species (ROS), which include hydrogen peroxide, hydroxyl radicals,

hypochlorite ion and peroxynitrites. (Figure 1-1) It is known that a particularly

destructive aspect of oxidative stress is due to the overproduction of these reactive

oxygen species, which causes an imbalance between the production of reactive oxygen

and a biological system's ability to readily detoxify the reactive intermediates or easily

repair the resulting damage. For example, the hydroxyl and hydroperoxide radicals in

Figure 1-1 the generation of reactive oxygen species (ROS) and the toxic effects to damage the cell


3

biological systems preferably attack polyunsaturated fatty acids, and the attack brings

about crosslinking and polymerization of the fatty acid structures. Oxidative stress has

been considered as a major cause of cellular injuries in a variety of clinical

abnormalities, especially prominent in neural diseases. In humans, oxidative stress is

involved in many diseases, such as atherosclerosis, Parkinson's disease and

Alzheimer's disease and it also has been shown to participate in a number of different

cancers as well as in the normal ageing process.

1.1.2 Superoxide Dismutases (SODs) and their Active Metal Center Structures

Normally cells are able to defend themselves against ROS damage through the

use of enzymes such as superoxide dismutases (SODs), a class of oxidoreductase

enzymes, which keep the concentration of superoxide radicals at low limits and

therefore play an important role in the defence against oxidative stress. In this reaction

the oxidation state of the metal cation oscillates between n and n+1. In mammals,

SODs are generally classified according to the metal species which acts as

redox-active center: SOD1 or Cu/ZnSOD and SOD3 or EC-SOD, which have Cu and

Zn in their catalytic center, while SOD2 or MnSOD has Mn in the catalytic center.

SOD1 is primarily cytoplasmic, SOD2 is restricted to mitochondria and SOD3 is

extracellular.[2] All anaerobic prokaryotes, if they possess SOD activity, will contain

FeSOD exclusively.[3, 4] Recently a new superoxide dismutase containing nickel

(NiSOD) was purified and suggested to represent a novel class of superoxide

dismutases on its own since no amino acid sequence homology is found in enzymes of

the two already existing classes.[5] The SOD-catalysed dismutation of superoxide may

be written with the following half-reactions:

M(n+1)+−SOD + O2− → Mn+ − SOD + O2

Mn+−SOD + O2− + 2H+ → M(n+1)+ − SOD + H2O2

where M = Cu (n=1); Mn (n=2); Fe (n=2); Ni (n=2)

These enzymes perform this catalytic cycle of dismutation with incredible efficiency,

the mammalian CuZn-SODs have been shown to possess catalytic rates in excess of 2


4

x 109 M-1 s-1, while the Mn and Fe SOD enzymes have been shown to function at rates

that are somewhat slower, approximately an order of magnitude slower depending on

the source of the enzyme.

Structure comparisons of several SODs of each type reported from PDB bank

show that the MnSOD and FeSOD groups are closely related to each other, whereas

the CuZnSODs appear to have evolved independently. The active site of the CuZnSOD

enzyme is shown in Figure 1-2, CuZnSOD has both a copper ion and a zinc ion

embedded in its structure. The metal ions in SOD1 and SOD3 are bridged by the

imidazole ring of residue His63 which acts as a ligand to both metals. The Cu(II) is

further coordinated to three histidine residues (44, 46 and 118) and a water molecule to

form a distorted square pyramidal geometry, while the Zn coordination is completed

by a further two histidines (69 and 78) and an aspartate (81) in a distorted tetrahedral

geometry.[6-7] There is structural evidence from crystallography and EXAFS that the

Cu–His61–Zn bridge in SOD1 is broken upon reduction to Cu(I), leaving an

approximate trigonal planar Cu coordination.[8]

Figure 1-2 The protein crystal structure of CuZn-SOD and the structure of its active site


5

Most crystal structures of MnSODs have been determined at room temperature

and have a five-coordinate, trigonal bipyramidal active site geometry. (Figure 1-3)

The active site manganese is located between the helical hairpin and the β sheet

structural elements. In the resting state, the active site metal ion is in the trivalent state,

Mn3+, which is coordinated by three N atoms from histidine residues (90, 145 and 232),

one O atom from Asp228 and one oxygen atom of OH– group (or a water molecule) to

a distorted trigonal bipyramidal environment. The two axial ligands are His90 and or

water, with the equatorial plane formed by His145, Asp228 and His232. The bond

lengths and angles show a high regularity in geometry, with the His90-N–Mn–OH– (or

water) angle (176O) very close to the ideal 180O, and the Mn only 0.09 Å out of the

equatorial plane.[9, 10, 11] However, the 1.55 Å resolution atomic coordinates of 100

K E. coli manganese superoxide dismutase revealed there is a sixth hydroxide ligand

from the electron density map (Mn-OH2(O) 2.42(0.01) Å). It means that at low

temperature the manganese also can act as six-coordinate octahedral active sites and

this sixth coordinate site maybe implicated in closing-off the active site.[12]

Figure 1-3 The protein crystal structure of Mn-SOD and the structure of its active site; Mn-His90(N) 2.19(0.03); Mn-His145(N) 2.19(0.02); Mn-Asp228(O) 2.04(0.04); Mn-His232(N) 2.19(0.01); Mn-OH(O) 2.19(0.02) Å


6

The Fe- and Mn-containing superoxide dismutases catalyze the same reaction

and have almost superimposable active sites. The coordination geometry at the Fe site

in FeSOD is also distorted trigonal bipyramidal, with axial ligands His43 and solvent

W171 (proposed to be OH–), and in-plane ligands His95, Asp171, and His199.[13]

(Figure 1-4) Moreover, reduction of crystals to the FeII state does not result in

significant changes in metal-ligand geometry. However, the positions of the distal

azide nitrogens are different in the FeSOD and MnSOD complexes. The geometries of

the FeIII, FeII, and FeIII -azide species suggest a reaction mechanism for superoxide

dismutation in which the metal alternates between five- and six-coordination.[14]

1.2 Development and Considerations of Synzymes as SOD Mimics

Although protective and beneficial roles of superoxide dismutase have been

demonstrated in a broad range of diseases both preclinically and clinically,[15] for

example, the most importantly, human clinical results with, Orgotein1 (bovine

CuZnSOD) showed promising results as a human therapy under acute and chronic

conditions associated with inflammation, including rheumatoid arthritis and

osteoarthritis as well as side effects (acute and chronic) associated with chemotherapy

and radiation therapy.[16, 17] However, in some situations such as a stroke or

Parkinson's disease, these native enzymes do not show efficacy because they can not

Figure 1-4 The protein crystal structure of Fe-SOD and the structure of its active site; Fe-His43(N) 2.18(0.02); Fe-His95(N) 2.14(0.02); Fe-Asp171(O) 1.92(0.03); Fe-His199(N) 2.10(0.02); Fe-W171(O) 2.0(0.03) Å


7

penetrate (because of their large size, MW ~30 KD) the blood brain barrier, moreover,

the non-human origin of these enzymes inevitably gave rise to a variety of

immunological problems.[18]

As a discipline, medicinal inorganic chemistry grew very fast over the last 40

years since the first discovery of the antitumor activity of cisplatin,

cis-[Pt(NH3)2Cl2].[19] To overcome many of these limitations from native SOD

enzymes, for example, their large sizes, the consequences of which are low cell

permeability, a short circulating half-life, antigenicity and high-manufacturing costs,

many research groups have been pursuing the possibility of rational design and

synthesis of low molecular weight catalysts like cis-[Pt(NH3)2Cl2] and developing such

SOD synzymes[20] as an approach to mimic the natural SOD enzymes’ function.

Compared with the native SOD enzymes, such SOD synzymes have many distinct

advantages as pharmaceutical agents because they have the ability to access

intercellular space, the lack of immunogenicity, a longer half-life in the blood (the

human enzymes are stable in vivo for only short periods, i.e., t1/2 only several minutes),

potential for oral delivery, and a lower cost of goods. In recent years tremendous

progress has been made in this area both in defining a role for such a synthetic enzyme

as a human pharmacological agent utilizing a number of animal models for disease and

in progressing toward development of actual drug candidates. This could allow the

synthetic superoxide dismutase mimetics to serve as pharmaceutical candidates in a

variety of diseases in which the native SOD enzyme was found to be effective. The

recent developments achieved in this active field can be summarized into the following

four classes along the ligand.[21] (Figure 1-5)


8

1.2.1. Manganese(III) Metalloporphyrins

Since the earliest report of SOD activity by a manganese porphyrin complex

was reported by Pasternack and co-workers nearly 30 years ago with the tetrakis

(4-N-methylpyridyl)porphine complex of MnIII,[22] manganese(III) metalloporphyrins

complexes have been identified as SOD mimics and studied by several groups. The

manganese moiety of the porphyrin based SOD functions in the dismutation reaction

with superoxide by successive reduction followed by oxidation changes in its valence

between MnIII and MnII, much like native SODs. Structure–activity relationships have

guided the development of Mn porphyrins with highly positive metal-centered redox

potentials of ≥ + 200 mV vs. NHE, which means that metalloporphyrins are a unique

class of stable catalytic antioxidants possessing a broad range of antioxidant capacities,

i.e. ability to scavenge reactive oxygen (ROS) and nitrogen species (RNS), such as

O2•−, ONOO−, CO3

•− and •NO.[23-26] Moreover, metalloporphyrins like MnIII

tetrakis(4-benzoic acid)porphyrin (MnTBAP) are also potent inhibitors of lipid

peroxidation, exerting a protective effect against some of the dentrimental effects

Figure 1-5 The widely studied four classes of synzymes as SOD mimics


9

associated with endotoxic shock.[27] Since these SODm scavenge other reactive

oxygen species including peroxynitrite, the efficacy of MnTBAP in these models

probably relates to its peroxynitrite-scavenging activity in addition to its

superoxide-scavenging activity.[28] The Aeolus Pharmaceuticals Company is

currently developing a series of these metalloporphyrins as a subsidiary of Incara

Pharmaceuticals.

1.2.2. Manganese(III) salen Complexes

Although the synthetic metal-salen complexes have been studied by chemists

for more than half a century, experimental and clinical applications of the chiral salen

derivatives have attracted attention only since the 1990s. In manganese salen

complexes, salen ligands bind Mn ions through four atoms. One of the unique features

of these compounds is that the metal center is coordinated to oxygen and nitrogen

atoms, which is in contrast to macrocycles and porphyrins where the metal is

coordinated to nitrogen atoms only. The coordination of Mn by four axial ligands

results in the formation of several possible valance states, which are thought to be

important in the scavenging of a wide variety of ROS and, thus, contribute to the

non-selective nature of this class of antioxidant. These MnIII salen complexes have

been reported to have two key antioxidant properties via the scavenging of O2•− and

H2O2.[29] As an SOD mimetic, the MnIII in the salen complex is reduced to MnII by the

superoxide anion and yields oxygen. The MnII is then re-oxidized to MnIII by another

superoxide anion and produces hydrogen peroxide. Among this class of complexes, the

EUK series is currently being developed by Eukarion to commercial therapeutic

applicability. In experimental and clinical applications, EUKs have been proved it to

protect cells from oxidative damage in several animal models including Alzheimer's

disease,[30] Parkinson's disease,[31] stroke,[32] motor neuron disease[33] and

excitotoxic neural injury.[34] These findings indicate that manganese-salen complexes

(Mn-salens), a group of low molecular weight, cell-permeable complexes, can be used

as SOD/catalase mimetics.


10

1.2.3. Nitroxide

Nitroxide also known as Aminoxyls, are chemical compounds containing the

tertiary amine (R3N+-O-) functional groups that are oxidized to form relatively stable

nitroxide radicals. Many of these compounds have been synthesized and described, but

it has only been during the past 15–20 years that many of the interesting biochemical

interactions were discovered and exploited for medical use. One of the unique features

of these compounds is that they are metal free, so it is possible to avoid the toxicity

from the metal as the synzyme. The mechanism underlying the biologic activity of

these compounds is related to their ability to react with superoxide (Eq 3 and Eq 4) like

the function as superoxide dismutase (SOD) mimics.[35, 36]

These unique characteristics suggested that nitroxides, such as 4-hydroxy-

2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against

ionizing radiation. Tempol is a water-soluble analogue of the spin label TEMPO,

which is one of the nitroxides and now widely employed in electron spin resonance

spectroscopy. Its high stab ability and low molecular weight (172g/mol) allows it to

cross biological membranes easily and work intracellular. In fact, there is now good

evidence that Tempol exerts beneficial effects in animal models of shock,

ischaemia–reperfusion injury, inflammation, hypertension, diabetes and endothelial

cell disfunction.[37] However, tempol also reduces the formation of hydroxyl

radicals[38] and attenuates the cytotoxic effects of hydrogen peroxide, which is

mediated by hydroxyl radicals, supporting its non-selectivity.[39]

R'N

RO O2 O2

H+ R'N

ROH

R'N

ROH O2

H+

H2O2

R'N

RO

3

4


11

1.2.4. Manganese(II) (pentaazamacrocyclic ligand)-Based Complexes

Among all these four classes of complexes that have been studied as potential

SOD mimetics, the most efficient synthetic SOD catalysts known to date are

seven-coordinate complexes of MnII with macrocyclic pentadentate chelates derived

from C-substituted pentaazacyclopentadecane [15]aneN5. Compared to the structures

of MnIII porphyrins and salen complexes, in this kind of complexes the central MnII

atom always is coordinated by five nitrogen atoms from the pentaazacyclopentadecane

[15]aneN5 ligand in the same plane and two solvent or anion ligands at the axial

position to form a distorted pentagon-bipyramidal MnN5O2 (5+2) coordination,

moreover, the central metal atom here is MnII instead of the MnIII. Riley and his

research group have reported many of these MnII (pentaazacyclopentadecane)

([15]aneN5) complexes possessing catalytic SOD activity by stopped-flow kinetic

analysis at [superoxide]/[Mn complex] > 100 and in vivo activity in a range of models

involving oxidative stress.[40,41,42] Moreover, this kind of agents can meet the four

major criteria which is critical for SODm: high SOD activity, high stability, selectivity

only for superoxide and in vivo efficacy.[43] These synthetic SODm are exemplified

by the prototypical complex M40403,[44] derived from the 15-membered macrocyclic

ligand 1,4,7,10,13-pentaazacyclopentadecane, containing the added

bis(cyclohexylpyridine) functionalities. M40403 is a stable, low molecular weight,

manganese-containing, non-peptidic molecule, possessing the function and catalytic

rate of native SOD enzymes, but with the advantage of being a much smaller molecule

(MW 483 vs 30,000 for the mimetic and native enzymes, respectively). Another

important advantage of these synthetic enzymes is that they do not possess the

bell-shaped curve that is a common characteristic to the native SOD enzyme. This

complex possesses a higher catalytic activity at pH = 7.4 than the native MnSOD

enzyme. In fact, its catalytic rate exceeds 1 x 109 M-1s-1, comparable to the native

Cu/Zn SOD enzymes.[45]


12

1.3 Characterization Arrays of SOD Activity

1.3.1 Indirect Assay

For any effort to develop true catalytically active SOD mimics, it is critical that

one be able to rapidly and quantitatively assay any putative mimic for such catalytic

SOD activity. Since the first discovery and activity of the SOD enzymes reported by

Fridovich and McCord using cytochrome c assay,[46] numerous indirect assays based

on this assay have been developed to measure SOD activity in which the amount of

superoxide is estimated by the reaction of superoxide with a redox indicator.[47, 48, 49]

In these assays, Cytochrome c or nitro blue tetrazolium (NBT) are the most often used

indicators in a system using xanthine/xanthine oxidase to generate steady-state low

levels of superoxide. Then a reporter molecule (e.g., ferricytochrome c) is reduced by

superoxide to give the reduced form of cytochrome c, which gives a spectral change.

Inhibition of this reduction of cytochrome c by scavenging or reducing the superoxide

concentration was taken as a measure of SOD activity. However, indirect assays can

give false positives for SOD activity if the agent being tested inhibits the production of

superoxide, oxidizes the reduced redox indicator, or reacts stoichiometrically (not

catalytically) with superoxide. The indirect assays do not discriminate among these

processes and in addition do not provide information regarding the mechanism of

action of putative SODm.[50]

1.3.2 Direct Arrays

The direct methods for measuring SOD activity fall into two categories:

stopped-flow kinetic analysis and pulse radiolysis.[51] Both of these methods allow

precise measurement of the rate of dismutation of superoxide by visualizing directly

the spectrophotometric decay of the superoxide anion in buffer solution. Pulse

radiolysis offers the possibility of determining kinetic parameters over a wide pH

range and the superoxide is generated by pulse irradiation of oxygen-saturated aqueous

solutions in the presence of formate. The reaction with the putative SOD mimic can


13

then be measured by observation of the spectrum of superoxide. However, the

radiolysis methods often rely on a steady-state generation of superoxide, where the

initial concentration of dissolved oxygen in water (which is about 100 μM under 1 atm

of air at 25°C) is the limiting factor for superoxide flux. Moreover, this technique is of

limited utility as a broadly applicable tool as it is not widely available to researchers,

due to the obvious problems associated with cost and equipment. This is unfortunate,

as a direct monitoring of superoxide decay affords the most reliable way of

ascertaining SOD activity and for probing mechanism via kinetics studies.

To overcome the limitations of indirect assays and pulse radiolysis, Riley et al.

have utilized stopped-flow kinetic analysis as a direct technique for monitoring

superoxide decays kinetics via the spectrophotometric signature of superoxide at 245

nm. From this type of analysis, an uncatalyzed decay of superoxide (second-order

kinetics) can be distinguished from a catalyzed decay of superoxide (first-order

kinetics) in the presence of a large excess of superoxide over the complex being

screened. A second order catalytic rate constant (kcat) can be obtained for an agent with

true catalytic SOD activity. This direct determination of a true kcat can be used to

directly compare and quantify the SOD activities of enzymes and/or mimetics under a

given set of conditions (e.g., defined pH and temperature). No direct comparisons can

be made between the kcat value and activity obtained from the cytochrome c assay or

other indirect assays. However, the stopped-flow procedure can only be used in

characterization for compounds which possess catalytic activities greater than kcat

>105.5 M-1 s-1 (at pH ≈ 7.4) due to the competing background second-order

self-dismutation of superoxide.[52]

1.3.3 Cyclic Voltammetry Method

In the evaluation of the SOD activity and antioxidant capacity, the Cyclic

Voltammetric (CV) method is a very good array to perform preliminary estimates and

check if they are in good range for superoxide dismutation.[53] The total SOD activity

of the sample is a function combining the biological oxidation potentials (OPs),


14

characterized by the E1/2 value, which reflect the specific redox power of this sample.

Because the catalytic disproportionation of O2·– requires redox reactions between

complex and superoxide, the sample’s redox potential should fall in the range between

−330 mV (vs. NHE at pH 7; O2/O2·-) and +890 mV (vs. NHE at pH 7; O2

·-/H2O2).[54]

1.4 Fullerene and its antioxidant ability

There is a great need for the discovery of new drugs and for the development of

targeted rational therapies. Due to the advantages of nanotechnology relevant to

developing therapeutics, for example, nanobiotechnology can create a better

understanding of cell biology because the molecules in the cell are organized in

nanometer-scale dimensions and they function as nanomachines. Approaches based on

the medical application of nanotechnology, nanomedicine, have been used recently for

target discovery and are now taking on a key role.[55, 56] Up to now, the

nanomaterials as drug candidates include dedrimers, fullerenes and nanobodies.[57]

As shown in Figure 1-6, Buckminsterfullerene (IUPAC name

(C60-Ih)[5,6]fullerene or C60) is a condensed ring aromatic carbon nanosphere, which

was discovered in 1985 by researchers at the University of Sussex and Rice University

Figure 1-6 structure of Buckyminsterfullerene (C60)


15

and named after Richard Buckminster Fuller.[58] Then their synthetic and property

studies has sparked off a real remarkable interdisciplinary research acitivity from many

different branches of science and engineering.[59] C60 has thirty C-C double bonds

which resembles a isocahedral formed with 12 pentagons and 20 hexagons, with a

carbon atom at the corners of each hexagon and a bond along each edge. The diameter

of a C60 molecule is about 7.2 Å. Although the carbon atoms in fullerene are all

conjugated, the superstructure is not a super aromatic compound and the double bonds

in fullerene are not all the same. The X-ray diffraction bond length values are 1.355 Å

for the [6,6] (between two hexagons) bond and 1.467 Å for the [5,6] (between a

hexagon and a pentagon) bond.[60]

Although the native C60 is insoluble in water because of its hydrophobic

non-polar nature, more and more water-soluble fullerene derivatives have been

synthesized via the carbon-carbon or carbon-nitrogen bond formation in fullerene

modifications during the two decades. It is because of one important feature of

fullerene molecules: they have numerous points of attachment, allowing for precise

grafting of active chemical groups in three-dimensional orientations.[61] This attribute

makes it possible to introduce hydrophilic functional groups to the molecule and get its

chemical modification, which allows positional control in matching fullerene

compounds to biological targets. Together with other attributes, namely the size of the

fullerene molecules, the redox potential and the relative inertness in biological systems,

it is possible to tailor requisite pharmacokinetic characteristics to fullerene-based

compounds.[62-65] The diverse reactivity of these water-soluble derivatives has been

extensively investigated to lead them promising candidates for many biomedical

applications, which include antioxidant and neuroprotective activities, antiviral and

antibacterial properties, DNA photocleavage, enzyme inhibition, anti-HIV activity,

antimicrobial activity and radiotracers.[66-72]

Because C60 with its thirty C=C bonds is a typical alkene that is lacking

electrons, in fact, C60 fullerene has 60 electrons but a closed shell configuration

requires 72 electrons, the outwardly vaulted surface of fullerenes and the alignment of

the electrons produced thereby cause a great reactivity to free radicals. It is a very


16

efficient free-radical scavenger, which labels this molecule as a “sponge of absorbing

free radical”.[73] Moreover, a previous report on the electron bandgap transition

energy of fullerenes found that, based on HOMO-LUMO electron orbital energy

calculations, the electron affinity of C60 can be explained qualitatively by considering

its numerous pyracylene units, which upon receiving 2 electrons could go from an

unstable 4n π-system to a stable aromatic 4n+2 π system.[74] C60 would be classified

as a metal, which can reversibly accept up to six electrons.[75, 76]

Up to today, there are already many papers reporting that water-soluble

fullerenes have excellent efficiency in eliminating superoxide radical species. The first

positive results were achieved using polyhydroxylated C60 named fullerenenols or

fullerols [C60(OH)n](Figure 1-7), which have shown to be excellent antioxidants and

can reduce apoptosis in cortical neurons cultures because of their high solubility and

their ability to cross the blood brain barriers. At the same time fullerols have also been

demonstrated to absorb many oxygen radicals per fullerene molecule and to reduce the

toxicity of free radical damage on neuronal tissue. Combination of the moderate

electron affinity and the allylic hydroxy functional group of water soluble fullerenols

makes them appropriate candidate for application such as free radical remover or water

soluble antioxidant in biological system.[77] It has been shown that polyhydroxylated

fullerene (C60(OH)n n = 6-24), fullerenol prevented hydrogen peroxide and cumene

hydroperox hydroperoxide-elicited damage in the hippocampus slices.[78]

OH n

fullerols [C60(OH)n]

O

HO OH

OOH

O

HO

O

O OH

HO O

C3 Figure 1-7 molecular structures of fulleroles and C3


17

Water-soluble fullerenol has shown excellent efficiency in eliminating superoxide

radical species and antioxidative activity of fullerenol was demonstrated to be better

than vitamin E in prevention of lipid peroxidation, induced by superoxide and

hydroxyl radicals.[79] Another widely studied fullerene is the C3 tris-malonyl-C60

derivative (Figure 1-7), which has been shown to be protective in cell culture and

animal models of injury, including bacterial sepsis, degeneration of dopaminergic

neurons in Parkinson’s disease, and nervous system ischemia. Moreover, Dugan et al.

offered evidences in support of catalytic superoxide dismutation mechanism instead of

direct radical attack on the C60 moiety, showing that the tris-malonyl-C60 derivative (C3)

could functionally replace mitochondrial manganese superoxide dismutase

(MnSOD).[72]

They proposed that targeted addition of selected substituents to the C60 sphere can

harness this metal-like property of C60 to allow specific regions of C60 to coordinate

and stabilize O2·—. (Figure 1-8) Semiempirical quantum-mechanical calculations were

carried out and predicted that regions around the malonic acid groups are the most

electron-deficient areas, C3 electrostatically drives superoxide anions toward these

areas on its surface, O2·— is then stabilized by hydrogen bonding with protons on the

carboxyl groups (or intercalated solvating H2O) until a second O2·— arrives to combine

with the original O2·—, allowing dismutation of O2

·— with the help of protons from the

carboxyl groups and/or local water molecules. These results show that fullerenes with

Figure 1-8 a) the proposed mechanism of superoxide dismutation by C3 b) the electron distribution on


18

their excellent radical-scavenging properties should could act as neuroprotectants and

should be able to be employed for the protection of biological membranes against

oxidative changes.

On the other hand, due to the unique 3D, nanoscale, core-shell molecular

architectures and the possibility of tuning their properties by changing the number,

chemical nature, and relative position of functional units within the branched structure,

dendrimers are also attractive material for the development of nanomedicines.

Moreover, specialized chemistry techniques allow precise control over the physical

and chemical properties of the dendrimers. For example, a dendrimer can be

water-soluble when its end-groups are hydrophilic groups, like carboxyl groups.

Weissleder and colleagues investigated if the multivalent attachment of small

molecules to nanoparticles can be used to increase specific binding affinity and to

reveal new biological properties of such nanomaterials.[80] This technique can enable

dendrimer modification of fullerene to get dendrimeric fullerene derivatives and impart

desirable biological properties of both of them.

1.5 Aims of this thesis

Since Paul Ehrlich proposed the concept of Structure-Activity-Relationship

(SAR) for the inorganic compound Arsphenamine at the beginning of the twentieth

century, as a discipline, medicinal inorganic chemistry has made much progress in the

development of inorganic complexes as therapeutic agents and diagnostics.[55] To

design a specific compound which can treat and cure a specific disease, studies should

be carried out to elucidate the compound’s mechanism of medicinal action and to

optimize and improve the compound’s physiological activity. In the pharmacological

use of SODm, the most active synthetic catalysts for superoxid disproportionation

known to date, are exactly the seven-coordinate complexes of MnII with macrocyclic

ligands derived from C-substituted pentaazacyclopentadecane [15]aneN5. Why and

how the seven-coordinate geometry of metal complexes engenders its remarkable

catalytic activity, exceeding that of the native mitochondrial MnSOD enzymes whose

coordination sphere of active metal centers is of different geometry? The aim of the


19

first part of this thesis is to experimentally clarify the mechanistic behaviour of already

proven, highly efficient seven-coordinate MnII and FeIII SOD mimics, to elucidate the

role of the coordination number of seven in the catalytic process and to perform

detailed kinetic studies on our new class of potentially SOD active seven-coordinate

manganese and iron complexes. This knowledge will be a key for the successful

designing and synthesizing catalytic drugs as a new generation of therapeutics.

On the other hand, due to the advantages of nanotechenology relevant to

developing therapeutics, approaches based on the medical application of

nanotechenology, nanomedicine, have been used recently for target discovery and are

now taking on a key role. Up to now, the nanomaterials as drug candidates include

dedrimers, fullerenes and nanobodies.[55] Is it possible to link fullerenes with

dendrimers by covalent bonds and apply the new dendrimeric fullerene derivatives as

superoxide dismutase mimics? The aim of the second part of this thesis is to

systematically study the mechanistic behaviour of reaction between fullerenes and

superoxide, to clarify the Structure-Activity-Relationship (SAR) between fullerenes

and SOD activity. This knowledge will be useful for the successful designing and

synthesizing new fullerene derivative catalytic drugs as a SOD mimic.

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Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity

25

Chapter 2

Seven-Coordinate Iron and Manganese Complexes with Acyclic

and Rigid Pentadentate Chelates and their Superoxide Dismutase

Activity

2.1 Abstract

The reactions of seven-coordinate [FeIII(dapsox)(H2O)2]ClO4·H2O (1),

[FeII(H2dapsox)(H2O)2](NO3)2·H2O (2) and [MnII(H2dapsox)(CH3OH)(H2O)]-

(ClO4)2(H2O) (3) complexes of the acyclic and rigid pentadentate H2dapsox ligand

(H2dapsox = 2,6-diacetylpyridine-bis(semioxamazide)) with superoxide have been

studied spectrophotometrically, electrochemically and by a sub-millisecond mixing

UV/vis stopped-flow in DMSO. The same studies were performed on the

seven-coordinate [MnII(Me2[15]pyridinaneN5)(H2O)2]Cl2·H2O (4) complex with the

flexible macrocyclic Me2[15]pyridinaneN5 ligand (Me2[15]pyridinaneN5 =

trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]-octadeca-1(18),14,16-triene)

which belongs to the class of proven superoxide dismutase (SOD) mimetics. The X-ray

crystal structures of 2, 3 and 4 were determined. All complexes possess pentagonal

bipyramidal geometry with the pentadentate ligand in the equatorial plane and solvent

molecules in the axial positions. The stopped-flow experiments in DMSO (0.06 % of

water) reveal that all four metal complexes catalyze the fast disproportionation of

superoxide under the applied experimental conditions, and the catalytic rate constants

are found to be (3.7 ± 0.5) x 106, (3.9 ± 0.5) x 106, (1.2 ± 0.3) x 107 and (5.3 ± 0.8) x


26

106 M-1 s-1 for 1, 2, 3 and 4, respectively. The cytochrome c McCord-Fridovich assay

in aqueous solution at pH = 7.8 resulted in the IC50 values (and corresponding kMcCF

constants) for 3 and 4, 0.013 ± 0.001 µM (1.9 ± 0.2 x 108 M-1 s-1) and 0.024 ± 0.001

µM (1.1 ± 0.3 x 108 M-1 s-1), respectively. IC50 values from NBT (nitroblue tetrazolium)

assay are found to be 6.45 ± 0.02 µM and 1.36 ± 0.03 µM for 1 and 4, respectively.

The data have been compared with those obtained by direct stopped-flow

measurements and discussed in terms of the side reactions that occur under the

conditions of indirect assays.

2.2 Introduction

Superoxide (O2•–) is the reactive radical anion formed following an one-electron

reduction of dioxygen during numerous oxidation reactions under normal conditions in

both living and non-living systems.[1] Since it is a very good reducing agent in the

anionic form, and a very good oxidant in the protonated form (pKa (HO2) = 4.69),

superoxide is potentially dangerous for all cellular macromolecules and can generate

other undesired reactive species.[1, 2] Its damaging effects lead to different

pathophysiological conditions that cause aging, pain, inflammatory disorders, serious

neuro-degenerative diseases and multiple types of cancer.[1b, 2b, 3] Therefore, the

concept of removing superoxide via rapid disproportionation, i.e. dismutation (Eq. 1),

has protective beneficial outcome in a large number of diseases caused by the

overproduction of superoxide radicals.[3a, 4]

2O2• – + 2H+ → H2O2 + O2 Eq. 1

Natural superoxide dismutase enzymes (SODs) catalyze reaction Eq. 1 and in

preclinical and clinical trials have shown promising therapeutic properties, although

they suffer as drug candidates primarily from immunogenic response.[2a, 3a, 4a, 5]

This calls for new types of free-radical inhibiting enzyme mimetics to be used as

pharmaceuticals. Stable low molecular weight metal complexes that can react with

superoxide and efficiently replicate the activity of the native SOD enzyme, have a


27

potential to become a new generation of drugs for the treatment of diseases of various

aetiologies.[2b, 3a, 4a, 5, 6]

Among many different complexes that have been studied as potential SOD

mimetics,[2b, 3a, 5, 6b, 7] the most efficient synthetic SOD catalysts known to date are

seven-coordinate complexes of MnII with macrocyclic pentadentate chelates derived

from C-substituted pentaazacyclopentadecane [15]aneN5 (Scheme 2-1).[2b, 3a, 4a, 5,

6a, 8]

Their catalytic rate constants were obtained by direct kinetic measurements, as the only

reliable method for quantitative assessment of activity,[9, 10] showing that the SOD

activity of these complexes can exceed that of the native mitochondrial MnSOD.[6a,

8g] At the same time these complexes are the first enzyme mimetics tested in humans.

In the case of the macrocyclic MnII mimetics (Scheme 2-1) it has been

postulated that the profound conformational rearrangements of the macrocyclic

pentadentates facilitate subsequent electron transfer and that the ligands with high

conformational flexibility can assist SOD activity.[8a, 8g] Seven-coordinate FeIII SOD

mimetics with the same macrocyclic chelate systems show a different catalytic

mechanism in which the aqua-hydroxo form of the complex, [FeIII(L)(OH)(H2O)]2+, is

the catalytically active species.[11] A drawback of these complexes is the low pKa

values of the two coordinated water molecules, which results in the formation of

inactive (inert) dihydroxo complexes at the physiological pH.[11] Therefore, the idea

is to design a chelate that will decrease the acidity of the iron center and so increase the

concentration of the catalytically active aqua-hydroxo species at the physiological pH

N

HN NH

HN NH

Mn2+

Cl

Cl

NHHN

NHHN

NH

M

Cl

Cl

Scheme 2-1


28

to promote an enhanced SOD activity. Since free iron ions are more toxic than

manganese ions,[5] it is important that the chelate will form a very stable complex and

prevent the release of iron ions. Despite this toxicity, complexes of FeIII would be

highly attractive as SOD mimetics due to their higher kinetic and thermodynamic

stability than MnII complexes.

Since we have shown that the conformational flexibility of the pentadentate

ligand is not a key requirement for the SOD activity of the seven-coordinate complexes,

due to the fact that in an interchange substitution mechanism (operating in the case of

these complexes)[12a] efficient formation of a real six-coordinate (with pseudo

octahedral geometry) intermediate is generally not required, we were interested in

additional experimental validation of such mechanistic paradigm. This has been

achieved by probing the reactivity of appropriate conformationally inflexible

complexes towards superoxide.

In this chapter we have synthesized and characterized seven-coordinate FeII (2)

and MnII (3) complexes of acyclic and rigid pentadentate H2dapsox (H2dapsox =

2,6-diacetylpyridine-bis(semioxamazide))[12b] that have several important features

regarding their potential SOD activity.

The reactivity of these two complexes and the previously reported FeIII complex (1) of

the same ligand[12c, 12d, 12e] (all of which have the structure shown in Scheme 2-2)

towards superoxide has been studied spectrophotometrically, electrochemically and by

a sub-millisecond mixing UV/vis stopped-flow in DMSO. Catalytic SOD activity of

N

N

NN

N

O

O

H 2 NO

H 2 NO

M

H 2 O

H 2 O

( H )

( H )

M = F e I I I , F e I I , M n I I

Scheme 2-2


29

the complexes with the acyclic and rigid H2dapsox ligand has been compared (under

the selected experimental conditions) with the reactivity of a MnII (4) complex with the

flexible pyridine derivative of the [15]aneN5 macrocycle (Me2[15]pyridinaneN5 =

trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo-[12.3.1]-octadeca-1(18),14,16-triene),

which belongs to the class of proven SOD-mimetics.[8e] The SOD activity of the

complexes has also been investigated in aqueous solution by applying indirect

cytochrome c and NBT (nitroblue tetrazolium) assay and the data have been compared

with those obtained by direct stopped-flow measurements and discussed in terms of the

side reactions that occurs under the conditions of indirect assays.

2.3 Experimental Section

Materials

All solid chemicals were of p.a. grade and used as received without any further

purification. [Zn(H2dapsox)(H2O)2]Cl2 used in the electrochemical measurements was

synthesized according to the published procedure.[13] HPLC grade DMSO containing

a controlled amount of water (0.06 % after mixing in stopped-flow cuvette) was used

for the complex solutions, and the water content was determined by Karl Fischer

titration. KO2 solutions were prepared according to following procedure,[14] 100mg of

KO2 are ground in a mortar containing 25mL of Bu4N·PF6 DMSO(HPLC) solution,

then the solution is filled into a crew capped glass which is packed in aluminum foil to

secure the solution from light. After being stirred for 15 minutes, this solution is

filtered thought a hydrophobic PTFE filter (20μm) and kept in dark to avoid

decomposition though irradiation. This kind of superoxide solution has only very small

decay in 2.5 hours if kept under darkness.(Figure 2-1)

Instrumentation and Measurements

Carlo Erba Elemental Analysers 1106 and 1108 were used for chemical analysis.

IR and UV/vis spectra were recorded on a Mattson FT IR 60 AR (KBr pellets) and a

Hewlett-Packard 8542A spectrophotometer, respectively.


30

Time-resolved UV-vis spectra were recorded on a modified Bio-Logic

stopped-flow module μSFM-20 (10 ms dead time) combined with a Huber CC90

thermostat and equipped with a J & M TIDAS high speed diode array spectrometer

with combined deuterium and tungsten lamp (200–1015 nm wavelength range). Isolast

O-rings were used for all sealing purposes to enable measurements in DMSO. The

spectrum of DMSO was used as a reference for all spectroscopic measurements. For

the rapid kinetic measurements the Bio-Logic stopped-flow module was upgraded to a

sub-millisecond mixing stopped-flow configuration by combining it with a

microcuvette accessory (with an optical path light of 0.8 mm) and a monochromator to

minimize the dead time of the instrument. Measurements with the FeII complex were

performed under an atmosphere of dry nitrogen. Data were analyzed using the

integrated Bio-Kine software version 4.23 and also the Specfit/32TM program. At least

ten kinetic runs were recorded under all conditions, and the reported rate constants

represent the mean values.

Cyclic voltammetry measurements have been carried out using an Autolab

instrument with PGSTAT 30 potentiostat. A conventional three-electrode arrangement

was employed consisting of a gold disk working electrode (geometric area: 0.07 cm2)

(Metrohm), a platinum wire auxiliary electrode (Metrohm) and the Ag(s)/AgCl(s) wire

as pseudo reference electrode, for the measurements in DMSO, or a Ag/AgCl, NaCl (3

M) (Metrohm) reference electrode, for the measurements in the aqueous solution (the

potentials vs NHE was calibrated by the Ag/AgCl, NaCl (3 M), potential (0.222 vs

NHE)). The measurements in DMSO were performed in the presence of 0.1 M

tetrabutylammonium hexafluorophosphate as supporting electrolyte, whereas the

measurements in aqueous solutions were done applying a 0.1 M NaClO4 supporting

electrolyte. All solutions without superoxide were thoroughly degassed with nitrogen

prior to beginning the experiments and during the measurements the nitrogen

atmosphere was kept. Measurements with superoxide were carried out by saturating

the solution with dry oxygen ([O2] = 2.1 mM).[15] The sample concentration was 0.5

mM. All experiments were performed at room temperature.


31

Note: It should be pointed out that for DMSO solutions only glass equipment and

Hamilton teflon valves can be used!

Safety Notes. Perchlorate salts of metal complexes with organic ligands are potentially

explosive. Only a small amount of material should be prepared and handled with great care.

Synthesis of [FeII(H2dapsox)(H2O)2](NO3)2·H2O (2)

2,6-diacetylpyridine (0.163g, 1 mmol) and semioxamazide (0.218 g, 2.1 mmol) were

mixed in 60 mL of a methanol/acetonitrile mixture (2:1) and warmed up to 65 oC. The

reaction mixture was refluxed for 2 h under an argon atmosphere. Fe(NO3)3·4H2O

(0.313 g, 1 mmol) was carefully added to the resulting white suspension and its color

changed to gray. After 1 hour of refluxing, water (10 mL) was added resulting in a

clear gray solution which was cooled down to room temperature and left standing for 5

hours. The nice block crystals were filtered off, washed with a small amount of

acetone and dried in air (yield: 0.395 g, 71%). IR data (KBr, cm–1): 3509s(NH),

3386s(H2O), 3129s, 2929m, 1715s, 1677s, 1614s, 1541m(amide C=O), 1382s (amide),

1156m (CH), 822m, 681m.Anal. calcd. for C13H21N9O13Fe: C, 27.53; H, 3.73; N,

22.22%. Found: C, 27.74; H, 3.12; N, 22.49%.

Synthesis of [MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2(H2O) (3)

2,6-diacetylpyridine (0.163g, 1 mmol) and semioxamazide (0.218 g, 2.1 mmol) were

added to 40 mL of methanol and the mixture was stirred at 55 oC for 1 h.

Mn(ClO4)2·4H2O (0.326 g, 1 mmol) was added into the resulting white suspension.

The solution color changed to yellow, while some of the white powder still left

undissolved. The addition of 50 mL of CH3CN resulted in a clear yellow solution. Four

days later, light yellow crystals were collected (yield: 0.393 g, 60%).. IR data (KBr,

cm–1): 3501s(NH), 3361s(H2O), 3247s, 1720m, 1677s, 1529m(amide C=O),

1388s(amide), 1337s, 1147m (CH) 1048w, 819m, 737m, 669m. Anal. calcd. for

C14H23N7O15Cl2Mn.: C, 25.69, H, 3.54, N, 14.99%. Found: C, 25.47, H, 3.24, N

14.71%.

Synthesis of [MnII(Me2[15]pyridinaneN5)(H2O)2]Cl2·H2O (4)

3,6-diazaoctane-1,8-diamine (1.46g, 1.0mmol) was added dropwise to a hot solution of

MnCl2·2H2O (1.61g, 1.0 mmol) and 2,6-diacetylpyridine (1.63 g, 1.0mmol) in water


32

(50 mL). After four hours of refluxing the reaction mixture was filtered and the deep

orange solution was allowed to cool down to room temperature. Deep orange crystals

of the compound [MnIIL(H2O)2]Cl2 (L =

2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]-octadeca-1(18),2,12,14,16-pentaen

e) was obtained and dried under vacuum at 70OC for three hours (2.29 g, 69% yield).

This complex (2.2 g, ca. 5 mmol) was dissolved in 40 mL of anhydrous EtOH, and the

flask was flushed with argon for a few minutes. NaBH4 (20 mmol, ca. 4 equiv/double

bond) was added to the orange solution in one portion, and the suspension was stirred

at room temperature under argon. Two hours later, the temperature was increased to 60

°C and the mixture was stirred for next 3 h. After cooling down to room temperature,

the solvent was removed from the pale yellow mixture. The residue was dissolved in

water (20 mL) and NaCl (4.5 g) was added. The aqueous solution was extracted with

CH2Cl2 (3 x 40 mL). The combined organic phases were dried (MgSO4) and filtered,

and the solvent was removed. The pale yellow solid was dissolved into 5 mL of water.

Colorless crystals suitable for X-ray structure analysis were obtained after two days

(yield: 0.86 g, 18 ％ calculated from 2,6-diacetylpyridine). IR data (KBr,

cm–1):3524s(NH), 3460s, 3300s(H2O), 3210s, 2911m, 2868s, 1628m, 1597m,

1579m(amide C=O), 1458s, 1381m(amide), 1126m, 1106s, 1009m, 1106s(CH),

1009m, 980s, 808m, 680m. Anal. calcd. for C15H33Cl2MnN5O3: C, 39.40; H, 7.27; N,

15.31. Found: C, 39.38; H, 7.31; N, 15.29.

X-ray Crystal Structure Determinations.

Data for 2 and 3 were collected at 100 K using a Bruker-Nonius KappaCCD

diffractometer (λ = 0.71073 Å, graphite monochromator), while data for 4 were

collected at room temperature using a Siemens P4 four circle diffractometer (λ =

0.71073 Å, graphite monochromator). All data sets were corrected for Lorentz and

polarization effects. Absorption effects were taken into account by semiempirical

methods using either multiple scans (SADABS)[16a] for 2 and 3 or the Psi-scan


33

technique[16b] for 4. The structures were solved by direct methods and refined using

full-matrix least-squares procedures on F2 (SHELXTL NT 6.12).[16c] The perchlorate

anion in 3 is disordered, two alternative positions have been refined resulting in

occupancies of 52.0(6) % for O11–O14 and 48.0(6) % for O11′–O14′, respectively.

With the exception of the hydrogen atoms of the two methyl groups in 4 which are in

calculated positions of optimized geometry, the positions of all other hydrogen atoms

in 2, 3 and 4 were derived from difference fourier maps.

The isotropic displacement parameters of all hydrogen atoms were tied to those of the

equivalent isotropic displacement parameters of their corresponding C, N or O carrier

atoms. Crystal data, data collection parameters and refinement details of the structure

determinations of complexes 2–4 are summarized in Table 2-1.

2 3 4 Empirical formula C13H21N9O13Fe C14H21Cl2N7O14Mn C15H33Cl2N5O3Mn Formula weight 567.24 637.22 457.30 Temperature (K) 100(2) 100(2) 295(2) Wavelength (Å) 0.71073 0.71073 0.71073 Crystal system Monoclinic Monoclinic Orthorhombic Space group P21/c P21/c Pbca a (Å) 8.0028(6) 14.632(1) 10.245(1) b (Å) 14.6046(5) 11.257(1) 19.031(1) c (Å) 18.226(2) 14.908(2) 22.756(1) β (°) 93.079(9) 99.148(6) 113.65(1) V (Å3) 2127.1(3) 2424.3(4) 4436.8(5) Z 4 4 8 F(000) 1168 1300 1928

ρ (Mg/m3) 1.771 1.746 1.369

μ (mm-1) 0.799 0.848 0.859 Data/restrains/parameter 5491 / 4 / 388 5346 / 17 / 443 4525 / 0 / 334 GooF 1.034 1.059 0.977 R1 (I ≥ 2 (　 I)) 0.0262 0.0357 0.0526 wR2 (all data) 0.0671 0.0845 0.1245

a R1 = ∑||Fo|-|Fc||/∑|Fo|, wR2 = [∑w(Fo2-Fc

2)2/∑w(Fo2)2]1/2

Table 2-1 Crystal data and structure refinement for 2 to 4


34

Indirect SOD assays

Cytochrome c assay

SOD activities of complexes were measured using standard McCord-Fridovich

assay[17] based on ferricytochrome c reduction with superoxide produced by

xanthine/xanthine oxidase. The assay was performed at 25°C in 3 mL of reaction

buffer (50 mM potassium phosphate buffer, pH = 7.8) containing ferricytochrome c

(10 μM), xanthine (100 μM), and an amount of xanthine oxidase such as to give a rate

of ΔOD550nm ≈ 0.02 min–1 (about 0.01 UmL–1) in the absence of a putative SOD

mimic. A reduction of ferricytochrome c was monitored at 550 nm. After 150 s,

different amounts of the putative SOD mimic were added. Rates were linear for at least

8 min. Both rates in the absence and in the presence of the complex were determined

for each concentration of complex added and plotted against it. The IC50 value

represents the concentration of putative-SOD mimic that induces a 50% inhibition of

the reduction of cytochrome c.

Reliability of McCord-Fridovich assay

To check that the tested compounds do not inhibit the production of superoxide

by xanthine oxidase, the rate of conversion of xanthine to urate (see below) was

determined by measuring the increase in absorbance at 290 nm over a 2-min period

with and without the tested compounds. To measure the rate of conversion of xanthine

to urate, xanthine oxidase (20 μL of 1 UmL–1 XO) was added to a solution of 50 mM

potassium phosphate buffer pH 7.8 containing xanthine (150 μM) at a final volume of

1.0 mL at 25 °C. Urate production was monitored at 290 nm.[18] No difference in the

slope was recorded with or without the putative SOD mimics. To exclude the

possibility of hydrogen peroxide interference with the assay,[10] and with intent to

avoid catalase addition (that can make the system more complex), catalase activity of

complexes was monitored as described previously.[18] No catalase-type activity of our

complexes was detected.

Modified NBT assay


35

To further probe SOD activity of our complexes, a modified NBT assay was

used.[7i,19a] In this essay an extensive excess of superoxide against catalyst is used. 1

mg of solid KO2 was added into 2 mL of 50 mM potassium phosphate buffer pH 7.8

containing putative SOD mimetic and after 2 min spectra are recorded. NBT reacts

with superoxide forming blue pigment formazan (λmax ≈ 580 nm (35000 M-1

cm-1)).[19b] The presence of complex caused concentration dependent inhibition of

formazan formation, as fallowed by absorbance change at 580 nm. The concentration

that causes 50 % of formation was indicated as IC50

2.4 Results and Discussion

2.4.1 Stability of Superoxide Anion in H2O-DMSO

As a reliable, simple and well-understood method for the synthesis and

introduction of superoxide ion to biological or biomimetic systems, potassium

superoxide has been widely used, especially in dimethyl sulfoxide. We always evaluate

samples’ SOD activity by directly studying the decay of O2·— with stopped-flow

procedures. Because all biological or biomimetic systems are in the vivo, monitoring

the formation and decay of O2·— in vitro is important to study the influence of various

biological substances which by their real interaction with O2·— in biological or

biomimetic systems. However, the life-time of O2·— in aqueous solutions is very short

and the reaction between O2·— and water can completed in few millisecond, in this

thesis, we always used a H2O-DMSO mixture as solvent instant of aqueous solutions,

because O2·— can be kept longer in this mixture system and it is helpful for us to catch

the reaction which is due to the catalysts and not to water.


36

We started our studies by investigating the effect H2O% on the decay of O2·— UV/Vis

spectrum. When 20% water DMSO is mixed with 2mM superoxide solution, there is

very big absorbance change at 270 nm, which corresponds to the decay of O2·—. The

spectral changes are separated into two parts, the first part (ΔA ≈ 0.3) is a very fast

reaction and is completed in less than 2 millisecond (Figure 2-1b); Then followed the

second part (ΔA ≈ 0.4), which finish about 250 seconds and can be explained as the

spontaneous dismutation of O2·—.

0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040

0.42

0.45

0.48

0.51

0.54

0.57

0.60

0.63

0.66

0.69

Abs

orba

nce(

270n

m)

Time, s

DMSO + SO 20% water DMSO + SO 1% water DMSO + SO

0 50 100 150 200 250 300 350 400

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abs

orba

nce(

270n

m)

Time, s

20% water 1% water 2% water 5% water

c d Figure2-1 c) the kinetic traces of reaction between different concentrations H2O with O2

·— at 270nm in 40ms d) the dismutation of O2

·— in different water percent DMSO solution

250 300 350 400 450 5000.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25Ab

sorb

ance

Wavelength(nm)

0.000 0.001 0.002 0.003 0.004 0.0050.40

0.45

0.50

0.55

0.60

0.65

0.70

Abs

orba

nce

(270

nm)

Time, s

a b

Figure 2-1 a) the UV/Vis spectra of O2·— in pure DMSO followed 2.5 hours b) the kinetic traces of

reaction between 20% water DMSO with O2·— (2mM) at 270nm in 5ms


37

When we decrease the percent of water in DMSO, kinetic traces of the second part

recorded at 270 nm for various water percents (from 20% to 1%) upon mixing

solutions of O2·— and H2O-DMSO in the stopped-flow instrument at 25OC, are shown

in Figure 2-1d, we found that whole absorbance change at 270 nm of this part inverse

to the water percents in DMSO, which means that in the first step there have more

superoxide decomposed if there is more water in system. However, if the water percent

is lower than 1%, the whole absorbance changes of second part for various water

percents are very similar and to the kinetic traces recorded in 40ms at 270 nm we can

not find clear decay of O2·— any more during this time. (Figure 2-1c) This fact means

the life-time of O2·— in this solution is longer than in aqueous solutions and it is long

enough for us to evaluate the SOD activity of samples To make sure the decay of O2·—

is due to the samples and not to the water in DMSO during this time, during the

following measurements, we adopt DMSO containing a controlled amount of water

(0.06%) as solvent to dissolve our sample complexes, moreover, we can buffer in this

solvent to get ideal pH.

2.4.2 Structures

The cationic [FeII(H2dapsox)(H2O)2]2+ of complex 2 (Figure 2-2) has a

pentagonal-bipyramidal (PBP) structure with neutral pentadentate H2dapsox ligand in

Figure 2-2 ORTEP view of [FeII(H2dapta)(H2O)2(NO3)]+ in the crystal of 2 drawn with thermal elliposide at 50% probability level, another nitride anion is omitted for clarity


38

the equatorial plane and two water molecules in axial positions. H2dapsox is

coordinated to the FeII center through the pyridine nitrogen, two imine nitrogens and

two oxygen atoms of hydrazide C=O groups. These five donor atoms are almost

perfectly coplanar, and the mean deviation from planarity is just 0.0137 Å. The central

FeII ion is only 0.0034(5) Å below this plane. The sum of four chelate angles and the

bite O1-Fe1-O3 angle is 360.01o, very close to 360° for an ideal planar structure. Two

axial water molecules (O5 and O6) complete PBP, forming an almost linear angle

(O5-Fe1-O6 = 178.88(4)o). The intraligand bond lengths suggest that the neutral

H2dapsox ligand is coordinated to the FeII center in a hydrazide >C═N––NH––C═O

form, different from the α–oxiazine >C═N––N═C––O- form of the deprotonated

dapsox2- ligand present in the PBP structure of the corresponding FeIII

[FeIII(dapsox)(H2O)2]+ complex.[12c]

Bond Distances (Å)

Fe(1)-O(5) 2.144(1) Fe(1)-O(6) 2.159(1)

Fe(1)-O(1) 2.179(1) Fe(1)-O(3) 2.195(1)

Fe(1)-N(1) 2.206(2) Fe(1)-N(2) 2.233(2)

Fe(1)-N(5) 2.218(2)

O(3)-C(12) 1.232(2) O(1)-C(8) 1.234(2)

N(6)-C(12) 1.343(2) N(3)-C(8) 1.346(2)

N(2)-N(3) 1.375(2) N(2)-C(6) 1.289(2)

N(5)-N(6) 1.372(2) N(5)-C(10) 1.287(2)

Bond Angles (deg)

O(5)-Fe(1)-O(6) 178.88(4) O(5)-Fe(1)-N(5) 91.96(4)

O(5)-Fe(1)-O(1) 88.10(4) O(6)-Fe(1)-N(5) 87.04(4)

O(6)-Fe(1)-O(1) 93.01(4) O(1)-Fe(1)-N(5) 148.91(4)

O(5)-Fe(1)-O(3) 91.79(4) O(3)-Fe(1)-N(5) 71.80(4)

O(6)-Fe(1)-O(3) 88.37(4) N(1)-Fe(1)-N(5) 70.15(4)

O(1)-Fe(1)-O(3) 77.12(3) O(5)-Fe(1)-N(2) 88.53(4)

O(5)-Fe(1)-N(1) 90.03(4) O(6)-Fe(1)-N(2) 91.92(4)

O(6)-Fe(1)-N(1) 89.16(4) O(1)-Fe(1)-N(2) 71.14(4)

O(1)-Fe(1)-N(1) 140.93(4) O(3)-Fe(1)-N(2) 148.23(4)

O(3)-Fe(1)-N(1) 141.95(4) N(1)-Fe(1)-N(2) 69.80(4)

N(5)-Fe(1)-N(2) 139.95(4)

Table 2-2 selected bond lengths (Å) and bond angles (deg) of 2


39

In the FeII complex the average C-N distance is a bit longer (1.316 Å), whereas

the average C-O (1.233 Å) and N-N (1.374 Å) distances are shorter than those found in

the FeIII complex (1.296, 1.280 and 1.392 Å, respectively). Despite the change in

metal-ion size and ligand charge, there is just a small increase in the average Fe-N

equatorial bond length from the FeIII to the corresponding FeII complex (ca. 0.022 Å).

The same was observed in the case of the macrocyclic iron PBP complexes, where the

small effect was explained in terms of the rigid nature of the cyclic ligands.[20]

However, the acyclic nature of our ligand suggests that the weak sensitivity of the

Fe-N bonds to the change in metal oxidation state is a more general feature of the

unsaturated segments of the pentadentate ligands. From another side, the average Fe-O

equatorial bond length is significantly longer in the FeII (2.187 Å) than in the FeIII

(2.056 Å) complex. The negative charge of the coordinated α–oxiazine oxygen atoms

additionally strengths the FeIII-O bond. The prominent elongation of the average

Fe-OH2 bond length from the FeIII (2.028 Å) to the FeII complex (2.152 Å) is observed,

confirming that the axial distances reflect changes in the ion size more readily than the

equatorial once.[21] Interestingly, the asymmetry in the two Fe-N(imine) bonds in

[FeII(H2dapsox)(H2O)2]2+ (0.015 Å) is somewhat more prominent than in

[FeIII(dapsox)(H2O)2]+ (0.007 Å). However, it is ca. two times smaller than in the case

of the very similar [FeII(H2dapsc)(H2O)(Cl)]+ complex (0.034 Å), where the difference

in these two bonds was rationalized in terms of the high-spin d6 configuration and the

Jahn-Teller effect in a PBP field.[21] The difference in the two equatorial Fe-O bonds

is identical (ca. 0.02 Å) in all three structures ([FeIII(dapsox)(H2O)2]+,

[FeII(H2dapsox)(H2O)2]2+ and [FeII(H2dapsc)(H2O)(Cl)]+), and is not affected by the

change in the iron oxidation state and charge of the oxygen atom. The average Fe-N

and Fe-O equatorial bonds in [FeII(H2dapsox)(H2O)2]2+ and [FeII(H2dapsc)(H2O)(Cl)]+

are almost identical. Even more, the average Fe-OH2 bond length in

[FeII(H2dapsox)(H2O)2]2+ and the corresponding bond length in

[FeII(H2dapsc)(H2O)(Cl)]+ are also identical. This shows that the axial coordination of

Cl- does not affect the bonds neither in the equatorial plane nor in its trans position.


40

The two terminal –NH2 groups are involved in intramolecular hydrogen bonds

with the metal-coordinated hydrazide oxygens (N4––H…O1 2.666(2) Å and N7––H…O3

2.714(2) Å; given are only the donor acceptor distances for the hydrogen bridges

discussed and complete details can be found in Table 2-3), similar to those observed in

the structure of [FeIII(dapsox)(H2O)2]+.[12c] The presence of the hydrogen atom on the

hydrazide nitrogen enables the formation of additional intramolecular hydrogen bonds

between the hydrazide nitrogen atoms N6 or N3 and the amide oxygen atoms O2 and O4

(N3––H…O2 2.793(2) Å and N6––H…O4 2.692(2) Å). Although hydrogen bond

interaction between the cationic complex and the NO3- counter anion is observed

(N7––H…O11 3.002(2) Å, N4––H…O11 3.486(2) Å) the ion pair association in this

Donor—H…Acceptor d(D-H) (Å) d(H···A) (Å) d(D···A) (Å) <(D-H···A) (O)

Intramolecular

N(4)—H(4A)…O(1) 0.87 2.30 2.6660 105

N(7)—H(7E)…O(3) 0.83 2.39 2.7143 104

N(6)—H(6N)…O(4) 0.82 2.41 2.6923 101

O(6)—H(6B)…O(12) 0.82 1.91 2.7258 170

N(4)—H(4A)…O(11) 0.87 2.64 3.4862 163

N(7)—H(7E)…O(11) 0.83 2.19 3.0016 164

Intermolecular

N(3)—H(3N)…O(7)(a) 0.86 1.97 2.8182 170

N(4)—H(4A)…O(13)(b) 0.87 2.56 3.0265 115

N(4)—H(4B)…O(13)(c) 0.85 2.09 2.9088 164

N(4)—H(4B)…O(4)(d) 0.85 2.58 3.0322 115

O(5)—H(5A)…O(22)(e) 0.84 1.91 2.7304 164

O(5)—H(5B)…O(21)(d) 0.83 1.93 2.7273 161

O(6)—H(6A)…O(23)(b) 0.82 1.98 2.7810 166

N(6)—H(6N)…O(12)(f) 0.82 2.37 3.0508 141

N(6)—H(6N)…O(2)(g) 0.82 2.45 2.9778 123

N(7)—H(7D)…O(6)(b) 0.83 2.45 3.0074 126

N(7)—H(7D)…O(7)(b) 0.83 2.29 2.9683 139

O(7)—H(7F)…O(2)(f) 0.83 1.93 2.7020 155

O(7)—H(7G)…O(4)(h) 0.80 2.02 2.7571 152

Table 2-3 Analysis of Potential Hydrogen Bonds of 2, (a) x,1/2-y,-1/2+z; (b) -1+x,y,z;(c) -x,1-y,-z;(d) -1+x,1/2-y,-1/2+z; (e) 1-x,-1/2+y,1/2-z; (g) x,1/2-y,1/2+z; (g) -x,-1/2+y,1/2-z;(h) 2-x,-1/2+y,1/2-z


41

structure is not so prominent as in the case of the interactions between ClO4- and

[FeIII(dapsox)(H2O)2]+, where ClO4- participates in an extensive hydrogen bond network.

Interestingly, this interaction between ClO4- and amide tails of the tweezer like cationic

complex is even observed in the MeCN solution of the FeIII, as well as FeII form of the

complex.[22]

The [MnII(H2dapsox)(CH3OH)(H2O)]2+ of complex 3 (Figure 2-3) has also the

PBP structure with the neutral pentadentate ligand coordinated in the equatorial plane

in a hydrazide >C═N––NH––C═O form. Thus, the intraligand bond lengths are almost

identical with those in the above discussed FeII complex. However, the Mn-N and

Mn-O bonds are all longer than the corresponding Fe-N and Fe-O bonds. The sum of

the chelate angles and the bite angle is 359.97°, which means that the five donor atoms

from H2dapsox form an ideal planar structure. In fact, the mean deviation of this plane

is just 0.0195 Å, with the central MnII ion little below this plane (the distance from the

plane is ca. 0.0412(8) Å).

In comparison with the similar structure of [MnII(H2dapsc)(H2O)(Cl)]+ [21] it

can be seen that even though the average Mn-N bond length is the same for both

structures (ca. 2.291 Å), the average Mn-O equatorial bond length is significantly

longer in our complex (2.291 vs 2.216 Å), showing that there is no general interrelation

between the equatorial distances as it was suggested. Even more, the difference

between two Mn-O equatorial bond lengths is very significant (0.135 Å), although

Figure 2-3 ORTEP view of [MnII(H2dapsox)(H2O)(CH3OH)(ClO4)]+ in the crystal of 3 drawn with thermal elliposide at 55% probability level, another perchroride anion is omitted for clarity.


42

such distortion is not expected for the spherically symmetrical high-spin d5 electronic

configuration. This shows that the intra- and intermolecular secondary interactions

within the crystal packing have an important influence on the symmetry of the

coordination sphere. As in the case of 2, the intramolecular hydrogen bonds are

observed between terminal –NH2 groups and the corresponding metal-coordinated

hydrazide oxygens (N4––H…O1 2.703(2) Å and N7––H…O3 2.763(2) Å) and between

the hydrazide nitrogen and amide oxygen atoms (N3––H…O2 2.749(2) Å and

N6––H…O4 2.630(2) Å).

As mentioned above, an interesting feature of the tweezer like complexes of H2dapsox

is their association with the anion by means of shape recognition, hydrogen bond

complementarity and charge assistance. The perchlorate anion is chelated by the

cationic [MnII(H2dapsox)(CH3OH)(H2O)]2+ complex via hydrogen bonds

(N4––H…O23 2.924(3) Å, N7––H…O24 3.136(3) Å and N7––H…O22 3.578(3) Å)

Bond Distances (Å)

Mn(1)-O(5) 2.152(2) Mn(1)-N(1) 2.287(2)

Mn(1)-O(6) 2.192(2) Mn(1)-N(2) 2.306(2)

Mn(1)-O(1) 2.224(4) Mn(1)-O(3) 2.359(2)

Mn(1)-N(5) 2.281(2)

O(1)-C(8) 1.242(3) O(3)-C(12) 1.227(3)

N(2)-C(6) 1.283(3) N(5)-C(10) 1.281(3)

N(2)-N(3) 1.379(2) N(5)-N(6) 1.371(2)

N(3)-C(8) 1.338(3) N(6)-C(12) 1.347(3)

Bond Angles (deg)

O(5)-Mn(1)-O(6) 171.94(7) N(5)-Mn(1)-N(1) 68.09(6)

O(5)-Mn(1)-O(1) 91.62(6) O(5)-Mn(1)-N(2) 95.90(7)

O(6)-Mn(1)-O(1) 90.87(6) O(6)-Mn(1)-N(2) 92.16(6)

O(5)-Mn(1)-N(5) 87.48(7) O(1)-Mn(1)-N(2) 69.78(6)

O(6)-Mn(1)-N(5) 86.89(6) N(5)-Mn(1)-N(2) 136.46(6)

O(1)-Mn(1)-N(5) 153.70(6) N(1)-Mn(1)-N(2) 68.39(6)

O(5)-Mn(1)-N(1) 93.00(7) O(5)-Mn(1)-O(3) 87.57(6)

O(6)-Mn(1)-N(1) 90.25(7) O(6)-Mn(1)-O(3) 85.05(6)

O(1)-Mn(1)-N(1) 138.17(6) O(1)-Mn(1)-O(3) 84.31(5)

N(5)-Mn(1)-O(3) 69.40(6)



43

closing the cavity of the complex and forming sort of a macrocyclic structure (Table

2-5)

Donor—H…Acceptor d(D-H) (Å) d(H···A) (Å) d(D···A) (Å) <(D-H···A) (O)

Intramolecular

N(4)—H(4C)…O(1) 0.89 2.39 2.7031 101

N(7)—H(7D)…O(3) 0.90 2.47 2.7629 100

N(6)—H(6C)…O(4) 0.89 2.22 2.6303 108

O(6)—H(6A)…O(3) 0.84 2.16 3.03 163

N(4)—H(4C)…O(23) 0.89 2.10 2.9237 154

N(7)—H(7D)…O(24) 0.90 2.26 3.1360 164

N(7)—H(7D)…O(22) 0.90 2.52 3.5776 155

Intermolecular

N(3)—H(3B)…O(4)(a) 0.89 2.04 2.9170 170

N(4)—H(4B)…O(21)(e) 0.89 2.16 3.0404 170

O(5)—H(5A)…O(24)(c) 0.87 1.92 2.7732 171

O(6)—H(6A)…O(14)(d) 0.84 2.02 2.7902 152

O(6)—H(6B)…O(11)(a) 0.84 1.92 2.7014 154

N(7)—H(7E)…O(2)(b) 0.89 2.03 2.8465 151

C(2)—H(2A)…O(13)(c) 0.91 2.39 3.2145 151

C(7)—H(7A)…O(4)(a) 0.96 2.44 3.1111 126

C(11)—H(11C)…O(13)(d) 0.95 2.50 3.0487 117

Table 2-5 Analysis of Potential Hydrogen Bonds of 3, (a) x,-1+y,z; (b) x,1+y,z; (c) x,1/2-y,1/2+z; (d) -x,-1/2+y,1/2-z; (e) 1-x,-y,-z

Figure 2-4 ORTEP view of [MnII(Me2[15]pyridinaneN5)(H2O)2]2+ in the crystal of 4 drawn with thermal elliposide at 25% probability level, chloride anion is omitted for clarity.


44

In order to compare the reactivity of our complexes with that of a proven SOD

catalyst under the selected experimental conditions, we have synthesized and

characterized the [MnII(Me2[15]pyridinaneN5)(H2O)2]2+ complex (4) with the

flexible pyridine derivative of the [15]aneN5 macrocycle, which belongs to the class of

proven SOD-mimetics.[8] Similar to other [Mn([15]aneN5)] type complexes,[8e, 8g]

[MnII(Me2[15]pyridinaneN5)(H2O)2]2+ exists in the seven-coordinate PBP geometry

(Figure 2-4), but crystallizes as a trans-diaqua instead of a trans-dichloro complex.

The Mn-N bond distances and bond angles (Table 2-6) are all quite similar to those

observed for other complexes of the same class. 4 crystallizes as a mixture of S,S- and

R,R-dimethyl enantiomers with a C-CH3 and N-H pattern on the macrocycle that

alternates as up-down-up-down. Consequently, the two sides of the macrocyclic plane

and the two axial coordination sites as well, are chemically equivalent. By way of

comparison with the analogous imine groups-containing complex,[23] the average

Mn-N distance in 4 is somewhat longer, since the double bond containing ligand has a

smaller cavity. The structure of the FeIII complex with the same ligand is known,[24]

but the ligand is present in its R,S-dimethyl diastereomer form where two methyl

groups are on the same side of the FeN5 plane.


45

2.4.3 Electrochemistry

The metal-centered redox potential is the most important criterion for the

complex to be a SOD mimetic, since the catalytic disproportionation of O2·– requires

redox reactions between complex and superoxide (Scheme 2-3).

Bond Distances (Å)

Mn(1)-O(2) 2.241(3) Mn(1)-N(3) 2.330(4)

Mn(1)-N(1) 2.278(3) Mn(1)-N(2) 2.343(3)

Mn(1)-O(1) 2.282(3) Mn(1)-N(5) 2.352(3)

Mn(1)-N(4) 2.320(3)

Bond Angles (deg)

O(2)-Mn(1)-N(1) 89.0(2) O(2)-Mn(1)-N(2) 86.0(2)

O(2)-Mn(1)-O(1) 173.4(2) N(1)-Mn(1)-N(2) 70.2(2)

N(1)-Mn(1)-O(1) 85.3(2) O(1)-Mn(1)-N(2) 89.0(2)

O(2)-Mn(1)-N(4) 83.7(2) N(4)-Mn(1)-N(2) 145.2(2)

N(1)-Mn(1)-N(4) 142.4(2) N(3)-Mn(1)-N(2) 74.7(2)

O(1)-Mn(1)-N(4) 102.9(2) O(2)-Mn(1)-N(5) 95.0(2)

O(2)-Mn(1)-N(3) 99.1(2) N(1)-Mn(1)-N(5) 70.3(2)

N(1)-Mn(1)-N(3) 143.3(2) O(1)-Mn(1)-N(5) 86.2(2)

O(1)-Mn(1)-N(3) 83.6(2) N(4)-Mn(1)-N(5) 73.7(2)

N(4)-Mn(1)-N(3) 74.3(2) N(3)-Mn(1)-N(5) 143.2(2)

N(2)-Mn(1)-N(5) 140.5(2)


Scheme 2-3 the possible mechanism of our seven coordinate Fe or Mn complexes for the decomposition of superoxide


46

The complex redox potential should fall between the redox potentials for the reduction

and oxidation of O2·–, viz. -0.16 and +0.89 V vs NHE, respectively.[25]

Aqueous solutions of [Fe(dapsox)(H2O)2]ClO4 (1) in the pH range 1 to 12

exhibit a reversible redox wave for the FeIII/FeII couple, and no complex

decomposition or dimerisation was observed.[26] Furthermore, in the pH range 1 - 10

the metal-centered redox potential for [Fe(dapsox)(H2O)2]ClO4 is in the range required

for the possible SOD activity. For the FeIII complexes with [15]aneN5 type of chelates

that are proven SOD catalysts, the redox potentials were not reported for the

physiological pH, at which these complexes exist as an equilibrium mixture of the

dihydroxo and aqua-hydroxo species.[11] At pH ~ 3 they have the redox potential in

the range of 0.35 – 0.45 V vs NHE. In comparison, our complex at pH = 3 and 7.8

shows reversible redox behavior at 0.34 V and 0.05 V vs NHE, respectively. It should

be noted that dapsox2- causes an increase in the pKa values of the coordinated water

molecules (pKa1 = 5.8 and pKa2 = 9.5),[12d, 26] which are very close to those of the

native FeIII-SOD enzyme (~5 and ~9).[27] Thus at the physiological pH, almost 100 %

of the complex is in the catalytically active aqua-hydroxo form.

The cyclic voltammogram for 1 in DMSO purged with nitrogen exhibits a

reversible couple at -0.13 V vs Ag/AgCl electrode (Figure 2-5a), or -0.11 vs NHE,

-2 .4 -2.2 -2 .0 -1.8 -1 .6 -1.4 -1 .2 -1 .0 -0.8 -0 .6 -0.4 -0 .2 0.0 0.2 0.4

-1.0x10-5

-8.0x10-6

-6.0x10-6

-4.0x10-6

-2.0x10-6

0 .0

2.0x10-6

4.0x10-6

6.0x10-6

+ e -O 2.- O 2

+ e -Fe II Fe III

- e -

Fe III- e -

Fe IIO 2O 2

.-

I, A

E (V ) vs Ag /A gC l

a b c d

Figure 2-5 Cyclic voltammogramms of a) 1 purged with nitrogen; b) 1 purged with dioxygen c) 1 purged with dioxygen d) pure DMSO purged with dioxygen. Conditions: [complex] = 0.5 x 10-3 M, [Bu4NBF4] =

0.1 M, T = 298 K, scan rates = 0.2 V/s.


47

obtained by calibration with ferrocene. The cyclic voltammogram in dioxygen

saturated DMSO in the scan range up to -0.4 V (Figure 2-5b) shows again reversible

redox wave for the FeIII/FeII couple at slightly more negative potential, -0.18 V, since

in the presence of oxygen it is more difficult to reduce FeIII. When the scan proceeds

toward more negative potentials (Figure 2-5c), after the complex is reduced to the FeII

species, molecular oxygen is reduced to superoxide at -0.82 V. When the scan is then

returned to 0.2 V, no corresponding anodic peak assigned to the oxidation of O2•- is

found, in contrast to the reversible redox behavior for dioxygen in DMSO solutions

(Figure 2-5d). The intensity of the anodic peak corresponding to the oxidation of FeII

is also significantly decreased. This suggests that the iron complex (starting from the

electrochemically generated FeII form) catalytically decomposes superoxide (the FeII

form, present in lower concentration than O2•-, consumes all of it). The superoxide

decomposition is also observed by applying much lower (catalytic) concentrations of

the complex.

Similar to the proven MnII seven-coordinate SOD mimetics with [15]aneN5 type

of chelates,[8b] 3 is stable in the pH range 6-10.5 and in methanol exhibits a reversible

redox potential at 0.8 V vs NHE.[8a, 8b] The redox behavior in aqueous solutions for

the macrocyclic manganese SOD mimetics was not reported. We measured the cyclic

voltammograms for 4 (Eox = 0.98 V and Ered = 0.35 V) and 3 (Eox = 0.64 V and Ered =

0.20 V) at pH = 7.8, and both complexes show similar behavior with large peak

separation.


48

Electrochemical measurements in dioxygen saturated DMSO (Figure 2-6) show,

as in the case of 1, that 3 can also catalytically decompose superoxide (disappearance

of the anodic peak assigned to the oxidation of O2•- in the presence of the MnII form of

the complex), remaining unchanged (appearance of reversible redox wave for the

MnII/MnIII couple at 0.65 V). In comparison, the proven SOD mimetic 4 upon reaction

with superoxide undergoes modification and in the scan range from 0 V to 1.2 V and

back to 0 V, three oxidation and reduction peaks appear.

Control electrochemical measurements of the [Zn(H2dapsox)(H2O)2]2+

complex in nitrogen and dioxygen saturated DMSO confirmed that H2dapsox was not

the redox active ligand (the zinc complex is electrochemically silent), and that in the

presence of its non-redox active metal complex electrochemically generated

superoxide was stable (the anodic peak assigned to the oxidation of O2•- does not

disappear in the presence of the zinc complex).

2.4.4 Reaction with superoxide in DMSO

We studied the reactions of 1, 2, 3 and 4 (Figure 2-7), with a large excess of

O2·– in DMSO containing a controlled amount of water (0.06 %), which was in excess

over the superoxide and complex concentrations. Water present in the DMSO solution

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

-5.0x10-5

-4.0x10-5

-3.0x10-5

-2.0x10-5

-1.0x10-5

0.0

1.0x10-5

2.0x10-5

3.0x10-5

4.0x10-5

5.0x10-5

+ e-O2

O2.-

Mn3+Mn2+ + e-Mn3+Mn2+ + e-

Mn3+Mn2+ - e-Mn3+Mn2+ - e-

i (A

)

E(V) vs Ag/AgCl

Figure 2-6 Cyclic voltammogramm of 3 purged with dioxygen. Conditions: [3] = 0.5 x 10-3 M, [Bu4NBF4] = 0.1 M, T = 298 K, in DMSO solution., scan rates = 0.2 V/s.


49

plays an important role and enables the catalytic decomposition of O2·–. Similar to what

was reported in the literature,[8a] under absolute water free conditions only a

stoichiometric reaction between O2·– and the complex could be observed and the

catalytic process was suppressed.

Time resolved UV/vis spectra (Figure 2-7) show that immediately after mixing

a superoxide solution with a complex solution, rapid decomposition of O2·– (decrease in

absorbance in the 240-330 nm range within the dead time of the stopped-flow

instrument) was observed. The products of superoxide disproportionation, O2 and H2O2,

were qualitatively detected in all four experiments.[28] In the case of 4, following fast

superoxide decomposition, the complex starts to decompose slowly and results in the

formation of a light brown colloid precipitate (presumably MnO2) after ~3 h. Three

hours after mixing with KO2, acid was added to the solutions of complexes 1, 2 and 3,

Figure 2-7 Time resolved UV/vis spectra recorded for the reaction of 5 x 10-5 M complex with 1 mM KO2 in DMSO at room temperature for 1 (a), 2 (b), 3 (c) and 4 (d). A: spectrum recorded (measurements in tandem cuvette) before mixing; B: first spectrum obtained after mixing (using a stopped-flow module) followed by spectra recorded at time intervals of 10 s (total observation time 2.5 h). Inset: control reaction without addition of the complex followed over 2.5 h.


50

which resulted in the recovery of the initial 1 and 3 complexes, respectively. This

demonstrates that our acyclic complexes are more stable than the macrocyclic complex

4 under the applied experimental conditions, which is in agreement with the

electrochemical observations.

The rapid process was quantified by following the corresponding absorbance

decrease at 270 nm in a series of stopped-flow measurements, in which the catalytic

concentration of the studied complexes was varied. Application of a microcuvette

accessory (which reduced the dead time of the instrument down to 0.4 ms) enabled

observation of the fast disappearance of the 270 nm absorption, which could best be

fitted as a first-order process to obtain the characteristic kobs (s-1) value. When

experiments were performed using the complex solutions with a higher amount of

water, it was not possible to quantify the corresponding rate constants since the higher

water contents caused a mixing problem on a short time scale.

In Figure 2-8 the obtained kobs values are reported as a function of the complex

concentration for our iron and manganese complexes, as well as for the proven SOD

mimetic. A good linear correlation between kobs and the complex concentration was

0.0 5.0x10-5 1.0x10-4 1.5x10-4 2.0x10-40

200

400

600

800

1000

1200

1400

-0,015 -0,010 -0,005 0,000 0,005 0,010 0,0150,34

0,36

0,38

0,40

0,42

0,44

0,46

0,48

Abs

orba

nce

time, s

-0,015 -0,010 -0,005 0,000 0,005 0,010 0,0150,34

0,36

0,38

0,40

0,42

0,44

0,46

0,48

Abs

orba

nce

time, s

-0,015 -0,010 -0,005 0,000 0,005 0,010 0,0150,34

0,36

0,38

0,40

0,42

0,44

0,46

0,48

Abs

orba

nce

time, s

k obs, s

-1

[complex], M

1 3 4 2

Figure 2-8 Plots of k

obs versus [complex] for the reaction between complexes and saturated KO2 in

DMSO solution at room temperature; inset: kinetic trace for [1] = 2 x 10-4 M and control reaction without addition of the complex obtained by using sub-millisecond mixing stopped-flow configuration.


51

observed for all studied complexes. From the slope of the plot of kobs vs catalyst

concentration the catalytic rate constants (kcat)[9, 10] were determined (Table 2-7).

The kcat values show that both iron complexes and the macrocyclic manganese

complex have almost the same catalytic activity, within the error limits, whereas 3 has

approximately two times higher activity. It should be stressed that it does not matter

whether the FeIII or FeII form of the complex is used, identical spectral changes

(Figure 2-7) and kinetic behavior (Figure 2-8) are observed upon reaction with

superoxide, which is consistent with the redox cycling of the complex during O2·–

decomposition (Scheme 2-3).

2.4.5 Reaction with superoxide in aqueous solution

To prove the reactivity of our complexes toward superoxide in aqueous buffer,

McCord-Fridovich assay (here referred as X/XO assay)[17] and modified nitroblue

tetrazolium (NBT)[7i, 19a] assays were used.

X/XO assay is based on kinetic competition for superoxide reaction between

oxidized cytochrome c and the complex showing SOD activity. The reduction of

ferricytochrome c was followed spectrophotometrically at 550 nm. Both, 3 and 4 were

complex k(cat) (M-1 s-1) IC50 ( M)　 kMcF (M-1 s-1)

MnSOD 0.005 ± 0.001 5.2 ± 0.2 x 108

1 (3.7 ± 0.5) x 106 - -

2 (3.9 ± 0.5) x 106 - -

3 (1.2 ± 0.3) x 107 0.013 ± 0.001 1.9 ± 0.2 x 108

4* (5.3 ± 0.8) x 106 0.024 ± 0.001 1.1 ± 0.3 x 108

*k(cat) = 1.0 x 107 M-1 s-1 was obtained in an stopped-flow experiment with DMSO/H2O = 1/188a-d, 9

Table 2-7 Catalytic rate constants and IC50 values obtained by using direct stopped-flow measurements in DMSO (0.06 % water) and indirect cytochrome c assay in aqueous solution (phosphate buffer pH = 7.8), respectively.


52

found to inhibit the reduction of the cytochrome c when injected into solution, as

shown in Figure 2-9a.

Inhibition percentages were measured for several complex concentrations (Figure 2-9b)

and IC50 values, calculated using the graphical method, are reported in Table 2-7.

Although used as a feature characterizing SOD activity of the complex, IC50 values

strongly depend on the concentration of the detector used and are thus not appropriate

for comparison with literature.[18] From the measured IC50 values, it is possible to

calculate a catalytic constant (kMcCF), which is independent of the detector

concentration. At the IC50 concentration, superoxide reacts at the same rate with the

detector and the putative SOD mimic. Then, kMcCF = kdetector·[detector]/IC50.[29]

On comparing the obtained kMcCF constants with those found in the literature,[6, 18,

30] 3 belongs to the very active SOD mimetics.

We also have observed that the catalytic rate constants obtained by using X/XO

assay (Table 2-7) are at least one order of magnitude higher then those obtained by

stopped-flow method. We found this to be due to the direct reaction between

complexes and cytochrome c. When higher complex concentrations than those which

cause nearly 100% inhibition were used, a re-oxidation of reduced cytochrome c was

observed, suggesting that the oxidized form of the complex (generated during the

catalytic cycle) acts as an oxidant of cytochrome c.[31]

Figure. 2-9 a) Kinetics of the reduction of ferricytochrome c (550 nm) without and with the putative SOD mimics. Reduction of ferricytochrome c (fallowed during 300 s) caused by addition of indicated concentrations of tested complexes and MnSOD (E. coli). b) Inhibition percentage as a function of concentration of 4 and 5: IC50 determination.


53

The iron complex exhibits an opposite effect, acting in its reduced form as a

reductant of cytochrome c, and increases the slope of cytochrome c reduction when

injected into solution.[31] (Figure 2-9a) Thus, the calculation of its IC50 and kMcCF

were not possible. To prove its activity in aqueous medium, we used a modified NBT

assay. Here, instead of xhanthine/xhantine oxidase system, KO2 was used as source of

superoxide. When 1 was present in solution, an inhibition of blue formazan formation

was observed in a concentration dependent manner. The concentration of 1 that caused

50 % inhibition of formazan formation (followed at 580 nm) was 6.45 uM. 4 caused

50% inhibition at the concentration of 1.36 uM, while 3 showed no effect. However,

we observed that 3 reacts with NBT itself, presumably forming a new complex that, if

the higher complex concentrations (> 1 x 10-3 M) were used, immediately precipitated

as a yellow powder. The solution of 1 (> 1 x 10-3 M) gets slightly milky in the presence

of NBT after one day, whereas no interaction between 4 and NBT was observed.

Interactions with the NBT indicator account for no detectable SOD activity of 3 and

somewhat lower activity of 1 than what would be expected based on the stopped-flow

measurements.

Although neither of the indirect methods we used proved to be reliable, they

show, in the manner utilized in literature,[6, 18, 30] that our complexes exhibit

substantial SOD activity in aqueous solutions as well.

2.5 Conclusions

Although it has been postulated in the literature that only seven-coordinate

complexes of macrocyclic ligands with prominent conformational flexibility could

possess SOD-activity,[8g, 32] our seven-coordinate iron and manganese complexes

with the acyclic and rigid H2dapsox ligand demonstrate ability for catalytic

decomposition of superoxide. Similar to what usually was found in the case of the

macrocyclic pentadentate ligands,[11] the manganese complex shows higher SOD

activity than the corresponding iron complex. However, higher stability of the iron

complex over a very wide pH range is an advantage in terms of a possible application.


54

The demonstrated SOD activity of the rigid seven-coordinate complexes is in

agreement with our recent findings that water release and formation of a six-coordinate

intermediate, requiring conformational rearrangement of the ligand, is not the

rate-limiting step in the overall inner-sphere catalytic SOD pathway of the proven

macrocyclic SOD mimetics.[12a] Furthermore, it also shows that conformational

flexibility of the pentadentate ligand is not the key factor assisting SOD activity, and

that the acyclic and rigid ligand systems can also be considered as structural motifs for

designing SOD mimetics. An additional advantage can be the fact that their syntheses

are more economic than the syntheses of macrocyclic ligands.

We have also shown that the indirect SOD assays, which are the mostly used

methods for demonstrating complex SOD activity, are not very reliable[10] and if, they

can be applied only upon considering possible cross reactions between indicator

substance and the studied complex in their different oxidation forms, in which they

may occur within the SOD catalytic cycle. The direct stopped-flow method, where the

high excess of superoxide over complex can be utilized, is a better probe for a complex

SOD activity even though it requires DMSO medium. Importantly, as it was stressed

by Sawyer et al., even closer relation between the kinetic measurements in aprotic

media than in bulk water can be drawn with the processes in mitochondria, which are

the major source of superoxide in the aerobic organisms, since aprotic media “may be

representative of a hydrophobic biological matrix”.[33] Under less protic conditions,

causing longer half-live of O2-, efficient superoxide decomposition is even more

desirable.

2.6 Refernces

[1] (a) I. Fridovich, J. Biol. Chem. 1989, 264, 7761-7764. (b) I. Fridovich, Ann.

N. Y. Acad. Sci. 1999, 893, 13-18.


55

[2] a) S. I. Liochev, I. Fridovich, IUBMB Life 1999, 48, 157-161. (b) D.

Salvemini, C. Muscoli, D. P. Riley, S. Cuzzocrea, Pulm. Pharmacol. Ther.

2002, 15, 439-447, and references therein.

[3] (a) C. Muscoli, S. Cuzzocrea, D. P. Riley, J. L. Zweier, C. Thiemermann,

Z.-Q. Wang, D. Salvemini, Br. J. Pharmacol. 2003, 140, 445–460, and

references therein. (b) E. R. Stadtman, Curr. Med. Chem. 2004, 11,

1105–1112.

[4] (a) D. Salvemini, D. P. Riley, S. Cuzzocrea, Nat. Rev. Drug Discovery 2002, 1,

367–374. (b) S. Cuzzocrea, C. Thiemermann, D. Salvemini, Curr. Med. Chem.

2004, 11, 1147–1162.

[5] D. P. Riley, Chem. Rev. 1999, 99, 2573–2587, and references therein.

[6] (a) D. Salvemini, Z.-Q. Wang, J. L. Zweier, A. Samouilov, H. Macarthur, T. P.


286, 304–306. (b) Z. Vujaskovic, I. Batinic-Haberle, Z. N. Rabbani, Q.-F.

Feng, S. K. Kang, I. Spasojevic, T. V.Samulski, I. Fridovich, M. W. Dewhirst,

M. S. Anscher, Free Radical Biol. Med. 2002, 33, 857–863, and references

therein. (c) W. E. Samlowski, R. Petersen, S. Cuzzocrea, H. Macarthur, D.

Burton, J. R. McGregor, D. Salvemini, Nat. Med. 2003, 9, 750-755. (d) A.

Okado-Matsumoto, I. Batinic-Haberle, I. Fridovich, Free Radical Biol. Med.

2004, 37, 401-410.

[7] (a) I. Batinic-Haberle, I. Spasojevic, P. Hambright, L. Benov, A. L. Crumbliss,

I. Fridovich, Inorg. Chem. 1999, 38, 4011 – 4022. (b) H. Ohtsu, Y. Shimazaki,

A. Odani, O. Yamauchi, W. Mori, S. Itoh, S. Fukuzumi, J. Am. Chem. Soc.

2000, 122, 5733 – 5741. (c) D. Li, S. Li, D. Yang, J. Yu, J. Huang, Y. Li, W.

Tang, Inorg. Chem. 2003, 42, 6071–6080. (d) Z. Durackova, J. Labuda, J.

Inorg. Biochem. 1995, 58, 297 – 303. (e) Z. Liao, D. Xiang, D. Li, F. Mei, F.

Yun, Synth. React. Inorg. Met.-Org. Chem. 1998, 28, 1327–1341. (f) I.

Spasojević, I. Batinić-Haberle, R. D. Stevens, P. Hambright, A. N. Thorpe, J.


56

Grodkowski, P. Neta, I. Fridovich, Inorg. Chem. 2001, 40, 726-739. (g) S.

Yamaguchi, A. Kumagai, Y. Funahashi, K. Jitsukawa, H. Masuda, Inorg.

Chem. 2003, 42, 7698-7700. (h) I. Batinić-Haberle, I. Spasojević, R. Stevens,

D. Hambright, P. Neta, A. Okado-Matsumoto, I. Fridovich, J. Chem. Soc.

Dalton Trans., 2004, 1696-1702. (i) J. Shearer, L. M. Long, Inorg. Chem.

2006, 45, 2358–2360. (j) S. Durot, F. Lambert, J.-P. Renault, C. Policar, Eur.

J. Inorg. Chem. 2005, 2789–2793.

[8] (a) D. P. Riley, R. H. Weiss, J. Am. Chem. Soc. 1994, 116, 387-388. (b) D. P.

Riley, S. L. Henke, P. J. Lennon, R. H. Weiss, W. L. Neumann, W. J. Rivers,

K. W. Aston, K. R. Sample, H. Rahman, C.-S. Ling, J.-J. Shieh, D. H. Busch,

W. Szulbinski, Inorg. Chem. 1996, 35, 5213-5231. (c) D. P. Riley, P. J.

Lennon, W. L. Neumann, R. H. Weiss, J. Am. Chem. Soc. 1997, 119,

6522-6528; d) D. P. Riley, S. L. Henke, P. J. Lennon, K. Aston, Inorg. Chem.

1999, 38, 1908-1917. (e) D. P. Riley, Adv. Supramol. Chem. 2000, 6, 217-244;

f) R. Krämer, Angew. Chem. 2000, 112, 4641-4642; Angew. Chem. Int. Ed.

2000, 39, 4469-4470. (g) K. Aston, N. Rath, A. Naik, U. Slomczynska, O. F.

Schall and D. P. Riley, Inorg. Chem. 2001, 40, 1779-1789.

[9] D. P. Riley, W. J. Rivers, R. H. Weiss, Anal. Biochem. 1991, 196, 344–349.


Ryanll, D. P. Riley, J. Biol. Chem. 1993, 268(31), 23049.

[11] D. Zhang, H. D. Busch, P. L. Lennon, R. H. Weiss, W. L. Neumann, D. P.

Riley, Inorg. Chem. 1998, 37, 956–963, and references therein.

[12] (a) A. Dees, A. Zahl, R. Puchta, N. J. R. van Eikema Hommes, F. W.

Heinemann, I. Ivanovic-Burmazovic, Inorg. Chem. 2007, 46, 2459-2470. (b)

K. Andjelkovic, I. Ivanovic, B. V. Prelesnik, V. M. Leovac, D. Poleti,

Polyhedron 1996, 15(24), 4361-4366. (c) K. Andjelkovic, A. Bacchi, G.

Pelizzi, D. Jeremic, I. Ivanovic-Burmazovic, J. Coord. Chem. 2002, 55,

1385–1392. (d) I. Ivanovic-Burmazovic, M. S. A. Hamza, R. van Eldik, Inorg.


57

Chem. 2002, 41, 5150-5161. (e) I. Ivanovic-Burmazovic, M. S. A. Hamza, R.

van Eldik, Inorg. Chem. 2006, 45, 1575-1584.

[13] M. Sumar, I. Ivanovic-Burmazovic, I. Hodzic, K. Andjelkovic, Synth. React.

Inorg. and Met.-Org. Chem. 2002, 32, 721-737.

[14] K. Duerr, B. P. Macpherson, R. Warratz, F. Hampel, F. Tuczek, M. Helmreich,

N. Jux, I. Ivanovic-Burmazovic, J. Am. Chem. Soc. 2007, 129, 4217-4228.

[15] S. V. Kryatov, E. V. Rybak-Akimova, S. Schindler, Chem. Rev. 2005, 105,

2175-2226.

[16] (a) SADABS, 2.06, Bruker-AXS, Inc., 2002, Madison, WI, U.S.A. (b) A. C. T.

North, D. C. Phillips, F. S. Mathews, Acta Cryst. 1968, A24, 351-359. (c)

SHELXTL NT 6.12, Bruker-AXS, Inc., 2002, Madison, WI, U.S.A.

[17] J. M. McCord, and I. Fridovich, J. Biol. Chem. 1969, 244, 6049-6055.

[18] C. Policar, S. Durot, F. Lambert, M. Cesario, F. Ramiandrasoa, I.

Morgenstern-Badarau, Eur. J. Inorg. Chem. 2001, 1807-1818.

[19] (a) S. Dutta, S. Padhye, F. Ahmed, F. Sarkar, Inorg. Chim. Acta. 2005, 358,

3617-3624. (b) B. H. J. Bielski, G. G. Shiue, S. Bajuk, J. Phys. Chem. 1980, 84,

830.

[20] M. G. B. Drew, O. A. Hamid bin, S. M. Nelson, J. Chem. Soc., Dalton Trans.

1976, 1394.

[21] G. J. Palenik, and D. W. Wester, Inorg. Chem. 1978, 17, 864-870.

[22] D. Sarauli, V. Popova, A. Zahl, R. Puchta, I. Ivanović-Burmazović, Inorg.

Chem., accepted.

[23] J.-S. Omar, R.-R. Daniel, J. R.-H. del María, E. S.-T. Martha, Z.-U. Rafael, J.

Chem. Soc., Dalton Trans. 1998, 1551–1556.

[24] M. G. B. Drew, D. A. Rice, S. B. Silong, Polyhedron 1983, 2, 1053-1056.


58

[25] D. M. Stanbury, Adv. Inorg. Chem. 1989, 33, 70–138.

[26] D. Sarauli, R. Meier, G.-F. Liu, I. Ivanovic-Burmazovic, R. van Eldik, Inorg.

Chem. 2005, 44, 7624-7633.

[27] M. S. Lah, M. M. Dixon, K. A. Pattridge, W. C. Stallings, J. A. Fee, M. L.

Ludwig, Biochemistry 1995, 34, 1646–1660.

[28] For the O2 detection see: Karlin, K. D.; Cruse, R. W.; Gultneh, Y.; Farooq, A.;

Hayes, J. C.; Zubieta, J. J. Am. Chem. Soc. 1987, 109, 2668-2679. For the

H2O2 detection a peroxide indicator paper suitable for the organic solvents

(QUANTOFIX-Peroxide 100) was used.

[29] J. Butler, W. H. Koppenol, and E. Margoliash, J. Biol. Chem. 1982, 257,

10747-10750.

[30] (a) Z.-R. Liao, X.-F. Zheng, B.-S. Luo, L.-R. Shen, D.-F. Li, H.-L. Liu, W.

Zhao, Polyhedron 2001, 20, 2813–2821. (b) M.-C. Rodriguez, I.

Morgenstern-Badarau, M. Cesario, J. Guilhem, B. Keita, and L. Nadjo, Inorg.

Chem. 1996, 35, 7804-7810. (c) P. J. F. Gauuan, M. P. Trova, L.

Gregor-Boros, S. B. Bocckino, J. D. Crapo, B. J. Day, Bioorg. Med. Chem.

2002, 3013-3021. (d) N. Kitajima, M. Osawa, N. Tamura, Y. Morooka, T.

Hirano, M. Hirobe, T. Nagano, Inorg. Chem. 1993, 32, 1879-1880. (e) D. F.

Xiang, X. S. Tan, Q. W. Hang, W. X. Tang, B.-M. Wu, T. C. W. Mak, Inorg.

Chim. Acta 1998, 277, 21-25. (f) I. Spasojevic, I. Batinic-Haberle, R. D.

Stevens, P. Hambright, A. N. Thorpe, J. Grodkowski, P. Neta, I. Fridovich,

Inorg. Chem. 2001, 40, 726-739. (g) R. Kachadourian, I. Batinic-Haberle, I.

Fridovich, Inorg. Chem. 1999, 38, 391-396.

[31] Detailed kinetic investigations are in progress.

[32] D. P. Riley, Adv. Inorg. Chem. 2007, 59, 233–263.


59

[33] D.-H. Chin, G., Jr. Chiericato, E. J., Jr. Nanni, D. T. Sawyer, J. Am. Chem.

Soc. 1982, 104, 1296-1299.


60

Chapter 3

Ligand Electronic Properties in the Control of Redox Behavior

and Reactivity toward Superoxide in Seven Coordinate

Manganese Complex

3.1 Abstract

In this chapter we have synthesized and characterized two new seven-coordinate

manganese complexes [Mn(Dcphp)(CH3OH)2](CH3OH)2 (5) and

[MnII(Daphp)(H2O)2](ClO4)2 (6). The complex 5 possesses two N-coordinated

hydrazido (amido) groups of dideprotonated Dcphp2-, whereas two imine nitrogen

atoms of the analogue hydrazone Daphp ligand are involved in N5O2 coordination

sphere of 6. These structural features enable for the first time investigations of ligand

electronic effects on the redox properties and SOD activity of the seven-coordinate

manganese complexes. To this goal the reactivity of 5 and 6 towards superoxide in

DMSO has been studied by electrochemical, spectrophotometrical and submillisecond

mixing UV/vis stopped-flow measurements. The results show that 5 has a quite low

redox potential which corresponds to its MnIII/MnII couple, and that it is capable of

removing the superoxide radical with a catalytic rate constant found to be 6.14 ± 0.08 x

106 M-1 s-1. On the other hand, the MnIII/MnII redox potential of 6 is at least for 1 V

higher than that of 5 and the complex 6 has no SOD activity.

Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex

61

The feasibility of geometric transition from seven-coordinate to six-coordinate

structure upon oxidation of the MnII complex to its MnIII form has been revealed by

DFT calculations in the case of both complexes. These studies demonstrates that the

energies required for the water dissociation (ΔE) and consequently formation of

six-coordinate geometries around the MnIII centers in the case of 5 and 6 are -1.1

kcal/mol and +7.9 kcal/mol, respectively, suggesting that the higher stability of

six-coordinated MnIII form of 5 can be related to its lower redox potential and ability to

dismutate superoxide. It seems that both electronic and structural properties of the

pentadentate lignads Dcphp2- and Daphp have the effect on the redox behaviour and

SOD activity of the studied seven-coordinate manganese complexes.

3.2 Introduction

Superoxide (O2•–) reactions with metal centers are of interest since they occur

within enzymatic catalises (superoxide dismutases, SOD, and superoxide reductases,

SOR)[1, 2] as well as undesired processes that might cause pathophysiological

conditions. Their understanding helps in conceiving whether and how metal complexes

can be used as pharmaceuticals for treatment of disease states caused by superoxide

overproduction and possible negative effects of the superoxide interactions with metal

centers under physiological conditions. Up to now the most active SOD mimetics are

seven-coordinate macrocyclic MnII complexes of pentaazacrowns[3, 4] which have

been considered as clinical candidates for a variety of inflammation conditions.[5] The

effects of the ligand substituents on the SOD activity of that class of complexes have

been extensively investigated, and it has been concluded that their prominent

conformational flexibility is the key property assisting in the high SOD-activity.[6, 7]

In the native MnSOD enzymes, which catalyze the disproportionation of O2·-

into hydrogen peroxide and molecular oxygen with a catalytic cycle involving both

electron and proton transfer, the manganese ion in the active site cycles between the +2

and +3 oxidation state. Therefore, a metal-centered redox potential of manganese

complexes is the most important criterion for the complex to be a SOD mimetic. At the


62

same time, MnIII, a d4 ion, prefers a distorted octahedral structure. Thus, the feasibility

of geometric transition from seven-coordinate to six-coordinate, when the MnII

complex is oxidized to MnIII, and the stability of the MnIII complex to ensure the

reversibility between its reduction and oxidation is also a very important criterion for

the complex to be a SOD mimetic.[8] For a special metal complex, the redox protential

of the central metal atom can be adjusted over a wide range by the nature and the

arrangement of the donor atoms around the metal bonding site and the nature and

position of the substituents.[9] Incorporation of amide groups into the chelating ligand

can substantially modulate the electronic structure of the central transition metal ion

and stabilize high oxidation states.[10, 11]

Recently (see Chapter 2) we have shown that the acyclic and a rigid

pentadentate ligand system (H2dapsox = 2, 6-diacetylpyridine-bis(semioxamazide))

can also be considered as structural motive that supports SOD-activity of iron and

manganese complexes.[12] The advantage of such type of complexes is that their

syntheses are much easier and with higher yields than the syntheses of macrocyclic

ones. To understand the effects of electronic properties of acyclic and more rigid

pentadentate ligands on redox and structural features of corresponding MnII complexes

and their reactivity towards O2•– in this paper we have synthesized and characterized

the seven-coordinate [Mn(Dcphp)(CH3OH)2](CH3OH)2 (5) (H2Dcphp =

Pyridine-2,6-biscarboxylic acid -bis((N′-2-pyridine-2-yl) hydrazide) and

[MnII(Daphp)(H2O)2](ClO4)2 (6) complexes (Daphp = 2,6-diacetylpyridine

bis(2-pyridylhydrazone) and studied their reaction with KO2 electrochemically,

spectrophotometrically and by a sub-millisecond mixing UV/vis stopped-flow in

DMSO, in a manner recently published by us.[12]


Materials and Instrumentation and Measurements see Chapter 2 Preparation of 2,6-bis(methoxycarbonyl)pyridine


63

1.5mL sulphuric acid was added into the solution of pyridine-2,6-dicarboxylic acid

(8.35g, 50mmol) in 60mL methanol, then this solution was heated to reflux for 20

hours. While cooling, the white crude product crystallizes, which was filtered off and

washed with ether. (9.25g, yield: 95%)

Preparation of H2Dcphp ligand

A solution of 2-Hydrazinopyridine (10.9 g, 100 mmol) in toluene (100 mL) was added

rapidly to a solution of 2,6-bis (methoxycarbonyl) pyridine (9.7 g, 50 mmol) in toluene

(150 mL) in a 500 mL round-bottomed flask. The suspension was then refluxed (oil

bath) and stirred for 24 h, in the end these substance were completely dissolved and

became clear solution. The heat was turned off and the solution was slowly cooled

down to the normal temperature and stirred overnight. During this time, some white

solid precipitate from the solution; which was collected and washed with diethyl ether

(ca. 3 x 10 mL) and dried under vacuum (17.3g, yield 49.6%); the second crop was

obtained from the initial filtrate by removing the solvent by rotary evaporation under

low pressure, the residue was redissolved in chloroform (120 mL) and washed with

water (3 × 50 mL). The chloroform solution was dried with anhydrous MgSO4 and

then filtered, after the solvent was removed by rotary evaporation under low pressure,

9.32g white powder was gotten (yield 26.7%). IR data (KBr, cm–1): 3206s(NH),

3186s(H2O), 2929, 1674, 1665 (CO), 1584, 1569, 1529, 1434, 1406 (NHCO), 1321,

1257(CH), 999, 775, 655 (py). Anal. calcd. for C17H15N7O2·H2O: C, 55.58; H, 4.66; N,

26.69%. Found: C, 56.04; H, 4.52; N, 26. 94%.

Synthesis of [MnII(Dcphp)(CH3OH)2](CH3OH)2 (5)

To a stirred suspension of ligand H2Dcphp (0.367 g, ca 1 mmol) in absolutely

methanol (50 mL) was added a methanol solution (10 mL) of MnCl2·2H2O (0.852 g, 1

mmol). After 1 h of stirring, the precipitate was almost dissolved. Then NaOCH3

(0.108g, 2 mmol) was carefully added into the solution under protect of nitrogen

atmosphere, right now the color of solution changed from light yellow to red. After

two hours reflux, the red solution was filtered and the filtrate was concentrated to ca.

30 mL on a rotary evaporator, then 0.5 g of NaClO4 was added to it. The solution was


64

kept in refrigerator for an overnight period, during which time shiny deep-yellow

crystals deposited. The product was filtered and washed with small amount of Acton.

Yield: 0.14 g (26.4%). IR data (KBr, cm–1): 3329s(NH), 3066s(H2O), 2363m, 1701s,

1613s, 1516m(amide C=O), 1341s (amide), 1191m (CH), 999m, 692m.Anal. calcd. for

H 5.51 C 47.55 N 18.48 O 18.10 Mn 10.36 C21H29N7O6Mn: C, 47.55; H, 5.51; N,

18.48%. Found: C, 47.04; H, 5.12; N, 18.49%.

Synthesis of [MnII(Daphp)(H2O)2](ClO4)2 (6)

2,6-diacetylpyridine (1.63g, 10 mmol) and 2-Hydrazinopyridine (2.18 g, 20 mmol)

were added to 50 mL of methanol and the mixture was stirred at 55oC for one hour. A

solution of Mn(ClO4)2·4H2O (3.26 g, 10 mmol) in 20mL methanol was dropwise added

into the resulting white suspension. The solution color changed to yellow, while some

of the white powder still left undissolved. The addition of 30 mL of CH3CN resulted in

a clear yellow solution. After 3 hours reflux the hot reaction mixture was filtered

and the dark yellow residue discarded. The deep orange solution was then

allowed to cool to room temperature and kept in refrigerator, rendering deep

orange crystals of the compound, suitable for X-ray diractometry. (yield: 3.67 g,

57.8%).. IR data (KBr, cm–1): 3290s(NH), 3198s(H2O), 3112s, 2362m, 1701w, 1687s,

1551m, 1341m(amide), 1279m, 1243m(CH) 1085w, 819m, 775m, 630m(py). Anal.

calcd. for C19H23Cl2N7O10Mn.: C, 35.92, H, 3.65, N, 15.43%. Found: C, 35.47, H, 3.64,

N 15.71%.

X-ray Crystal Structure Determinations.

Data for 5, 6 were collected at 100 K using a Bruker-Nonius KappaCCD

diffractometer (λ = 0.71073 Å, graphite monochromator). Crystal data, data collection

parameters and refinement details of the structure determinations of complexes 5 and 6

are summarized in Table 3-1.


65

Complex 5 6

Empirical formula C21H29N7O6Mn C19H23Cl2N7O10Mn

Formula weight 530.45 635.28

Temperature (K) 100(2) K 100(2)

Wavelength (Å) 0.71073 0.71073

Crystal system Monoclinic Monoclinic

space group C2/c C2/c

a (Å) 25.251(2) 10.9681(8)

b (Å) 7.6250(5) 31.293(2)

c (Å) 27.574(2) 7.9353(5)

α(°) 90 90

β (°) 113.044(5) 108.492(5)

γ (°) 90 90

V (Å3) 4885.4(6) 2583.0(3)

Z 8 4

ρ (Mg/m3) 1.442 1.634

Absorption coefficient 0.591 0.785

F(000) 2216 1300

Crystal size (mm) 0.25 x 0.10 x 0.07 mm 0.28 x 0.19 x 0.12

Theta range for data

collection.

3.21 to 27.10 deg. 3.41 to 28.00

Limiting indices -32<=h<=32, -9<=k<=9,

-35<=l<=35

-14<=h<=14, -41<=k<=41,

-9<=l<=10

Reflections collected /

unique

55979 / 5386 [Rint = 0.0550] 30122 / 3121 [Rint = 0.0682]

Max. and min. transmission 1.000 and 0.877 1.000 and 0.827

Data/restraints / parameters 5386 / 3 / 403 3121 / 24 / 206

GooF 1.124 1.038

R1 0.0408 0.0404

R2 (all data) 0.0916 0.0819

Largest diff. peak and hole (e

Å-3)

0.343 and -0.351 0.333 and -0.353

Table 3-1 Crystal data and structure refinement for 5-6


66


3.4.1 Structure

Bond Distances (Å)

Mn1-O4 2.1946(16) N2-C6 1.309(3) Mn1-N5 2.2655(18) N2-N3 1.385(2)

Mn1-N2 2.2693(18) N5-C12 1.307(3)

Mn1-O3 2.2823(16) N5-N6 1.390(2)

Mn1-N1 2.3319(18) O1-C6 1.290(3)

Mn1-N7 2.3600(18) O2-C12 1.281(3)

Mn1-N4 2.4676(19)

Bond Angles (deg)

O4-Mn1-O3 177.69(6) N5-Mn1-N7 69.18(6)

O4-Mn1-N5 86.11(6) N2-Mn1-N7 152.39(6)

O4-Mn1-N2 100.03(6) O3-Mn1-N7 83.71(6)

N5-Mn1-N2 134.93(7) N1-Mn1-N7 135.25(6)

N5-Mn1-O3 94.12(6) O4-Mn1-N4 83.02(6)

N2-Mn1-O3 81.44(6) N5-Mn1-N4 156.52(6)

O4-Mn1-N1 94.10(6) N2-Mn1-N4 67.77(6)

N5-Mn1-N1 67.65(6) O3-Mn1-N4 95.95(6)

N2-Mn1-N1 67.39(6) N1-Mn1-N4 133.76(6)

O3-Mn1-N1 88.11(6) N7-Mn1-N4 90.90(6)

O4-Mn1-N7 94.23(6)

Figure 3-1 ORTEP view of [MnII(Dcphp)(CH3OH)2)] in the crystal of 5 drawn with thermal elliposide at 35% probability level, other two methanol molecules are omitted for clarity and selected bond length (Å )and angle (O).


67

X-ray strucures (Table 3-1) of both complexes show (Figure 3-1 and Figure

3-2) a distorted pentagon-bipyramidal geometry around MnII with five nitrogen atoms

of the pentadentate ligands in the equatorial positions and two oxygen atoms of

methanol or water as axial donors. The Mn–N bond lengths in complex 5 range from

2.266(2) to 2.468(2) Å and give rise to four five-membered chelate rings, adjacent

N–Mn–N bond angles range from 67.39(6) to 69.18(6)o, which are slightly smaller

than the ideal value of 72O for a pentagonal-bipyramidal arrangement. (Figure 3-1)

Moreover, the disjunctive N4-Mn1-N7 angle is 90.90(6)o and the dihedral angle

between disadjacent five-membered rings [N5–N6–C13–N7–Mn1] and

N2–N3–C7–N4–Mn1] is 22.7O, which indicates the Dcphp2- ligand has a helical

conformation around the metal center and five N donor atoms from the pentachelate

ligand are not completely in the same plane (N2(+0.2528) and N7(+0.2796) up of the

plane, N4(-0.2742) and N5(-0.1989) off of the plane), and the mean deviation from

plane is 0.1775 Å. Compared to the structure of 5, the environment around the MnII

center in complex 6 (Figure 3-2) is very similar, the adjacent N–Mn–N bond angles

range from 67.57(4) to 68.47(6) o and the disjunct N4-Mn1-N7 angle is 91.73(9)o,

which is bigger than the corresponding angle of 5. The dihedral angle between

disadjacent five-membered rings is 24.8O, which means that these five N donors atoms

from Daphp ligand are also not completely in the same plane and the helical mode of

their coordination is more prominent than in the case of 5.


68

Bond Distances (Å)

Mn1-O1a 2.2131(16) Mn1-N4 2.3276(18)Mn1-O1 2.2131(16) Mn1-N4a 2.3276(18) Mn1-N1 2.305(2) N2-C6 1.290(3) Mn1-N2a 2.3230(18) N2-N3 1.354(2) Mn1-N2 2.3230(17)

Bond Angles (deg) O1a-Mn1-O1 169.88(9) O1-Mn1-N4 92.26(6)

O1a-Mn1-N1 95.06(4) N1-Mn1-N4 134.14(4)

O1-Mn1-N1 95.06(4) N2a-Mn1-N4 153.95(6)

O1a-Mn1-N2a 83.32(6) N2-Mn1-N4 68.47(6)

O1-Mn1-N2a 100.59(6) O1a-Mn1-N4a 92.26(6)

N1-Mn1-N2 67.57(4) O1-Mn1-N4a 80.66(6)

O1a-Mn1-N2 100.59(6) N1-Mn1-N4a 134.14(4)

O1-Mn1-N2 83.32(6) N2a-Mn1-N4a 68.47(6)

N1-Mn1-N2 67.57(4) N2-Mn1-N4a 153.95(6)

N2a-Mn1-N2 135.14(9) N4-Mn1-N4a 91.73(9)

O1a-Mn1-N4 80.66(6)

Figure 3-2 ORTEP view of [MnII(Daphp)(H2O)2]2+ in the crystal of 6 drawn with thermal elliposide at 50% probability level, two perchrorate ions are omitted for clarity and selected bond length (Å )and angle (O)..


69

Compared to the structure of the free H2Dcphp ligand (Figure 3-3), the

geometry of the hydrazide groups in 5 changes substantially upon its coordination, the

average C––Nhydrazide bond distance decreases from 1.34 to 1.30 Å and the average

C––Oamide increases from 1.235 to 1.294 Å. The average Mn1—Nhydrazide bond length

(2.267 Å) is significantly shorter than the recompensing average Mn1––Npyridine bond

length (2.385 Å). These structural features are result of the ligand negative charge and

its coordination in a hybrid form of the deprotonated hydrazide ––HN––N-––C═O and

α–oxiazine ––HN––N═C––O- resonance structures.(Scheme 3-1) The asymmetry in

the two Mn—Nterminal pyridine bonds in the crystal structure of 5 is prominent, which is

not typical for high-spin d5 electronic configurations. The intra- and especially

intermolecular hydrogen bonds (N6––H…O2d 2.902 Å d = 1-x, y, 1/2-z) within the

crystal packing are responsible for this asymmetry, similar to what has been observed

in the structure of [MnII(H2dapsox)(CH3OH)(H2O)]2+.[See chapter 2] The lack of the

carbonyl groups in Daphp makes it less acidic, resulting in its coordination in the

Bond Distances (Å)

O(1)-C(6) 1.2263(19) N(5)-N(6) 1.3889(18)

O(2)-C(12) 1.2368(19) N(5)-H(5N) 0.8867

N(2)-C(6) 1.347(2) N(5)-C(12) 1.333(2) N(2)-N(3) 1.3869(18) N(6)-C(13) 1.373(2)

N(2)-H(2N) 0.9113 N(6)-H(6N) 0.8875

N(3)-C(7) 1.369(2) C(5)-C(6) 1.507(2)

N(3)-H(3N) 0.9150 C(1)-C(12) 1.507(2)

Figure 3-3 ORTEP view of structure in the crystal of H2DcpHp ligand drawn with thermal elliposide at 30% probability level and selected bonds lengths (Å).


70

neutral hydrazone form, which is reflected in a longer average Mn1-Nimine bond length

and general higher symmetry in the Mn-N bond distances in the structure of 6

compared to those in 5.

3.4.2 Electrochemistry

The cyclic voltammogram of 5 in DMSO (Figure 3-4a) purged with nitrogen

exhibits a quasi-reversible MnIII/MnII couple at 0.372 V and irreversible reduction

process at -0.325 V vs Ag/AgCl, which is confirmed to be ligand centered by

measuring the cyclic voltammogram of free H2Dcphp on the same conditions.

0.0 0.2 0.4 0.6

-6.0x10-6

-3.0x10-6

0.0

3.0x10-6

6.0x10-6

9.0x10-6

300 400 500 600

0.2

0.4

0.6

0.8

1.0

Abso

rban

ce

Wavelength (nm)

i/A

E/V vs Ag/AgCl0.0 0.2 0.4 0.6

-6.0x10-6

-3.0x10-6

0.0

3.0x10-6

6.0x10-6

9.0x10-6

300 400 500 600

0.2

0.4

0.6

0.8

1.0

Abso

rban

ce

Wavelength (nm)

i/A

E/V vs Ag/AgCl

-1.5 -1.2 -0.9 -0.6 -0.3 0.0 0.3 0.6 0.9-6.0x10-5

-4.0x10-5

-2.0x10-5

0.0

2.0x10-5

4.0x10-5

6.0x10-5

i / A

E/V vs Ag/AgCl

A B C

a b

Figure 3-4 Cyclic voltammogramms of: a) 5 purged with nitrogen (inset: spectroelectrochemical oxidation of 5 at 0.5 V vs Ag/AgCl); 5 purged with nitrogen (A), oxygen (B), and pure DMSO purged with oxygen (C). Conditions: [5] = 0.5 x 10-3 M, [Bu4NPF6] = 0.1 M, T = 298 K, scan rates = 0.5 V/s

N O

HN

O

NHNHHN

N N

- 2H+ N O

N

O

NNHHN

N N

N O

N

O

NNHHN

N N

III

Scheme 3-1 The deprotonated process of H2Dcphp and resonance structures of the coordinated fragment


71

However, under the same experimental conditions 6 is electrochemically silent in the

scan range from -1 to 1.2 V (Figure 3-5b). These results clearly demonstrate the effect

of the deprotonated amide (in our case hydrazide) group present in the ligand on the

redox behavior of the manganese center. A strong σ-donor ability of the negatively

charged hydrazido nitrogen in the first coordination sphere of manganese significantly

decreases the MnIII/MnII redox potential by stabilizing the MnIII form of the complex,

which was characterized by spectroelectrochemical measurements (λmax = 360-390 nm,

inset in Figure 3-4a). Alternating measurements at 0.5 V and 0 V vs Ag/AgCl show

that 5 can be oxidized and reduced in a quantitatively reversible manner (Figure 3-5a).

In iron chemistry it is known that one amido nitrogen as a donor atom within TPA

(tris-(2-pyridylmethyl)amine) type ligands stabilizes the Fe3+ oxidation state in

six-coordinate geometry for about 1 V.[13] However in the seven-coordinate geometry

two deprotonated amid groups of the macrocyclic ligand stabilize the Fe3+ oxidation

state only for about 0.3 V.[14] An effect of the amide group on the redox behavior of

manganese complexes is less explored, and for the seven-coordinate complexes has not

been reported. It has been qualitatively demonstrated that bound carboxamido nitrogen

to the six-coordinate Mn center makes the complex more sensitive to the oxidation in

comparison to the analogous Schiff base complex.[15] Our results show for the first

time that in the seven-coordinate geometry coordination of two hydrazido nitrogens

instead of two imine nitogens stabilizes the MnIII oxidation state by at least 0.8 V.


72

Cyclic voltammetry measurements[12] were also performed in oxygen saturated

DMSO ([O2] = 2.1 mM)[16] in the scan range from 1 to -1.5 V (Figure 3-4b), and

when the scan proceeds toward more negative potentials, superoxide is generated by

oxygen reduction. When the scan is returned toward positive potentials no anodic peak

assigned to the oxidation of O2•- is found, suggesting that 5 present in solution

(experiments were performed with [5] = 1 mM and 0.1 mM) decomposes superoxide.

The same experiment was performed in the presence of free ligand and the

disappearance of the anodic peak assigned to the oxidation of O2•- was not observed.

Consequently, decomposition of O2•- in the presence of 5 is related to the metal

centered redox process. The 6 complex does not affect the superoxide reoxidation in

the same type of experiments. (Figure 3-5b)

3.4.3 Reactions with Superoxide in DMSO

The reaction of 5 with a large excess of KO2 in DMSO containing a controlled

amount of water (0.06 %) was followed by time resolved UV/vis spectroscopy, and the

rapid decomposition of O2·– was quantified by following the corresponding absorbance

decrease at 270 nm (best fitted as a first-order process) in a series of a sub-millisecond

300 400 500 600

0.2

0.4

0.6

0.8

1.0

Abs

orba

nce

Wavelength (nm)

reduction at 0 V vs Ag/AgCl

-3 -2 -1 0 1

-8.0x10-5

-6.0x10-5

-4.0x10-5

-2.0x10-5

0.0

2.0x10-5

4.0x10-5

i/A

E / V vs Ag / AgCl

6 purged with N2 pure DMSO + Air 6 purged with air

a b Figure 3-5 a) Spectroelectrochemical reduction of 5 at 0 V vs Ag/AgCl b) Cyclic voltammogramms of 6 purged with nitrogen, pure DMSO purged with oxygen and 6 purged with oxygen,. Conditions: [6] = 0.5 x 10-3 M, [Bu4NPF6] = 0.1 M, T = 298 K, scan rates = 0.5 V/s.


73

mixing stopped-flow measurements, in which the catalytic concentration of the studied

complexes was varied.[12]

A good linear correlation between corresponding kobs and the complex

concentration was observed, and from the slope of the plot of kobs vs. complex

concentration the catalytic rate constant (kcat)[17] was determined to be 6.1 ± 0.7 x 106

M-1s-1. (Figure 3-6) The products of superoxide disproportionation, O2 and H2O2, were

qualitatively detected.[12] Upon superoxide decomposition the complex remains in

solution in the MnIII form (λmax = 360-390 nm, inset in Figure 3-6), similar to what

was observed in the case of SOD active [MnII(H2dapsox)(CH3OH)(H2O)]2+.[12] As

expected from the electrochemical behavior of 6, its mixing with excess of KO2 in

DMSO does not cause rapid decay of the absorbance characteristic for superoxide.

Complex 5 Mn(H2dapsox)* MnII(pyaneN5)*

E1/2, V vs Ag/AgCl 0.37 0.66 0.80

kcat, s-1 M-1 (6.1 ± 0.7) x 106 (1.2 ± 0.3) x 107 (5.3 ± 0.8) x 106

*obtained under the same experimental conditions as in the present work.6

Table 3-2. E1/2 and SOD activities of three seven-coordinate Mn complexes.

0.0 2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-5 1.0x10-40

100

200

300

400

500

600

700

300 400 500 600 700 8000.0

0.3

0.6

0.9

1.2

1.5

1.8

B

A

Abs

orba

nce

Wavelength (nm)

k obs, s

-1

[5], M

Figure 3-6 Plots of kobs of decay at 270 nm versus [5]. Conditions: [O2•-] = 2 mM, 25OC in DMSO. Inset:

UV/vis spectra recorded for the reaction of 5 x 10-5 M of 5 with 1 mM KO2 in DMSO at room temperature. A: spectrum recorded before mixing; B: final spectrum obtained after KO2 decomposition.


74

Presented results reveal that 5 catalyzes the fast disproportionation of superoxide

under the applied experimental conditions, whereas 6 does not show SOD activity.

This can be explained by a high redox potential of 6, which does not fall between the

redox potentials for the reduction and oxidation of O2·– under the applied experimental

conditions. However, for the MnII complexes with redox potentials within that window

a correlation between their E1/2 and SOD activities (see Table 3-2 for the

seven-coordinate Mn complexes) is more complex. It seems that for the complexes

with lower E1/2, reduction of the MnIII form is a rate limiting step in the catalysis,

whereas for the SOD mimetics with higher E1/2, the catalytic cycle is a MnII oxidation

limited process. Data reported in the literature for the SOD active MnIII porphyrins[18]

and Mn complexes based on N-centered ligands[19] follow such a trend.

3.4.4 Modelling via DFT Calculations:

To probe whether the ability of 5 and 6 to form six-coordinate complexes in

their Mn2+ and Mn3+ oxidation states, upon release of a coordinated solvent molecule,

has an influence on their reactivity towards O2•–, in a manner that was reported for the

- H2O

+2.9 kcal

- H2O

-1.1 kcal

[MnIIDcphp(H2O)2]0 [MnIIDcphp(H2O)]0

[MnIIIDcphp(H2O)2]+ [MnIIIDcphp(H2O)]+

Figure 3-7 Calculated (B3LYP(CPCM)/LANL2DZp//B3LYP/LANL2DZp + ZPE (HF/LANL2MB)) water dissociation energy (ΔE) of [MnIIDcphp(H2O)2]0 and [MnIIIDcphp(H2O)2]+


75

macrocyclic MnII SOD mimetics,[12, 19-21] we have performed DFT calculations,[22]

the energies required for the solvent[23] dissociation were calculated to be +2.9 and

-1.1 kcal/mol for the MnII and MnIII forms of 5, respectively. These small energy

differences between seven- and six-coordinate geometries of both oxidation states,

suggest that solvent dissociation and formation of the six-coordinate intermediate are

not crucial for the complex SOD activity, in agreement with our recent findings.[12, 24]

In the case of 6 the solvent dissociation energies for its MnII and MnIII species are +1.7

and +7.9 kcal/mol, respectively. The later value indicates that formation of

six-coordinate MnIII form of 6 is significantly less favourable than in the case of 5.

This structural feature together with the electronic properties of Daphp might

contribute to the low stability of Mn3+ oxidation state of 6 and consequently its high

E1/2. Interestingly, in the case of macrocyclic MnII SOD mimetics, ligand structural

features do not affect their redox potential, but have an influence on their SOD activity.

It still remains to be seen to what extent the ligand structural and electronic features

can control the SOD activity of seven-coordinate manganese complexes by effecting

the mechanism of their substitution processes. Studies along these lines are in progress.

- H2O

+ 1.7 kcal

- H2O

+ 7.9 kcal

[MnIIDaphp(H2O)2]2+ [MnIIDaphp(H2O)]2+

[MnIIIDaphp(H2O)2]3+ [MnIIIDaphp(H2O)]3+

Figure 3-8 Calculated (B3LYP(CPCM)/LANL2DZp//B3LYP/LANL2DZp + ZPE (HF/LANL2MB)) water dissociation energy (ΔE) of [MnIIDaphp(H2O)2]2+ and [MnIIIDaphp(H2O)2]3+


76

3.5 Conclusions

In conclusion, both electronic and structural features of the acyclic N5

pentadentate ligands have a strong impact on the reactivity of seven-coordinate MnII

complexes towards superoxide. The deprotonated amide (hydrazide) groups with the

strong electron-donating and σ-donor ability in the first coordination sphere around the

manganese center in 5, significantly decrease MnIII/MnII redox potential by stabilizing

MnIII form of the complex. From the structural point of view, both MnII and MnIII

forms of 5 are almost equally stable as six- and seven-coordinate structures, which

supports the reversibility of the oxidation and reduction processes in which 5 is

involved. These two factors (electronic and structural) promote SOD activity of 5. The

complex 6, although structurally very similar to 5 in the Mn2+ oxidation state, does not

have ability to catalytically decompose superoxide. This is due to the fact that the

neutral N5 hydrazone ligand having the π-acceptor abilities strongly destabilizes the

MnIII form of 6, leading to the very high MnIII/MnII redox potential which does not fall

between the redox potentials for the reduction and oxidation of O2·– under the applied

experimental conditions. The relatively high energy required for the transformation of

the seven-coordinate MnIII structure to the corresponding six-coordinate geometry is

also an additional destabilizing factor of the MnIII form of 6.

3.6 References

[1] I. Fridovich, Ann. N. Y. Acad. Sci., 1999, 893, 13.

[2] J. P. Emerson, E. D. Coulter, D. E. Cabelli, R. S. Phillips, Jr., D. M. Kurtz,

Biochemistry, 2002, 41, 4348-4357.

[3] D. Salvemini, Z.-Q. Wang, J. L. Zweier, A. Samouilov, H. Macarthur, T. P.


286, 304.

[4] D. P. Riley, Chem. Rev. 1999, 99, 2573.


Wang, D. Salvemini, Br. J. Pharmacol. 2003, 140, 445.


77

[6] K. Aston, N. Rath, A. Naik, U. Slomczynska, O. F. Schall and D. P. Riley,

Inorg. Chem., 2001, 40, 1779.

[7] D. P. Riley, Adv. Inorg. Chem., 2007, 59, 233.

[8] D. P. Riley, S. L. Henke, P. J. Lennon, W. L. Neumann, K. Aston, Inorg.

Chem., 1999, 38(8), 1908.

[9] J. A. Streeky, D. Pillsbury, and D. H. Busch, Inorg. Chem. 1980, 19,

3148-3159.

[10] Collins, T. J. Acc. Chem. Res. 1994, 27, 279-285.

[11] Margerum, D. W. Pure Appl. Chem. 1983, 55, 23-34.

[12] For the experimental procedure regarding, electrochemical,

spectrophotometrical and kinetic measurements see: G.-F. Liu, M. Filipovic, F.

W. Heinemann and I. Ivanović-Burmazović, Inorg. Chem., 2007, 46, 8825

[13] J. M. Rowland, M. Olmstead, P. K. Mascharak, Inorg. Chem., 2001, 40, 2810.

In this reference, the complex with and those without the amide group, that

have been taken for a comparison, do not have the same number of

coordinated pyridine nitrogens, which can also account for a significant

difference in their redox potentials.

[14] I. V. Korendovych, R. J. Staples, W. M. Reiff, E. V. Rybak-Akimova, Inorg.

Chem., 2004, 43, 3930.

[15] K. Ghosh, A. A. Eroy-Reveles, B. Avila, T. R. Holman, M. M. Olmstead, P. K.

Mascharak, Inorg. Chem., 2004, 43, 2988.

[16] S. V. Kryatov, E. V. Rybak-Akimova, S. Schindler, Chem. Rev. 2005, 105,

2175.


Ryanll and D. P. Riley, J. Biol. Chem., 1993, 268(31), 23049.

[18] I. Batinic-Haberle, I. Spasojevic, R. D. Stevens, B. Bondurant, A.

Okado-Matsumoto, I. Fridovich, I. Vujaskovic, M. W. Dewhirst, Dalton

Transactions, 2006, 4, 617.

[19] S. Durot, C. Policar, F. Cisnetti, F. Lambert, J.-P. Renault, G. Pelosi, G. Blain,

H. Korri-Youssoufi and J.-P. Mahy, Eur. J. Inorg. Chem. 2005, 3513.


78

[20] K. Aston, N. Rath, A. Naik, U. Slomczynska, O. F. Schall and D. P. Riley,

Inorg. Chem., 2001, 40, 1779.

[21] Y. Che, B. R. Brooks, D. P. Riley, A. J. H. Reaka, G. R. Marshall, Chem. Bio.

& Drug Design, 2007, 69(2), 99.

[22] (B3LYP(CPCM)/LANL2DZp//B3LYP/LANL2DZp + ZPE

(HF/LANL2MB)).

[23] Usual for this type of calculations, for the simplicity, water was considered as

a coordinated solvent.

[24] A. Dees, A. Zahl, R. Puchta, N. J. R. van Eikema Hommes, F. W. Heinemann,

I. Ivanovic-Burmazovic, Inorg. Chem. 2007, 46, 2459.

Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes

79

Chapter 4

Structural Features in Control of Reactivity toward Superoxide

in Manganese and Iron Complexes

4.1 Abstract

In this chapter we report the synthesis and characterisation of a new

eight-coordinate manganese(II) complex 7 and a seven-coordinate FeIII dimer complex

8. The SOD activity of both complexes has been evaluated by electrochemical,

spectrophotometrical and submillisecond mixing UV/vis stopped-flow measurements,

which were carried out in DMSO solutions. The results show that these two complexes

are not able to catalytically decompose superoxide. The midpoint potential that

corresponds to the quasireversible MnIII/MnII couple of 7 is found to be 0.7 V vs

Ag/AgCl. Although this redox potential is in the range between the redox potentials for

oxidation and reduction of superoxide, the complex 7 can not dismutate superoxide

due to the fact that it does not have a free coordination site for superoxide binding to

the manganese center. The seven-coordinate FeIII dimer 8 is also redox active and

exhibits two reductions at –0.39 V and -0.57 V and two reversible oxidations at 0.32 V

and 0.49 V vs Ag/AgCl, despite its redox activity 8 is not SOD active. This illustrates

once more that the complex structural properties are equally important factors in

determining the efficiency of complex catalyzed superoxide decomposition.


80

4.2 Introduction As a functional SOD mimcs a number of seven coordinate manganese

complexes have been designed and synthesized with special ligand systems defining

the coordination number and geometry in high-spin manganese(II) complexes via

ligand steric and electronic effects.[1, 2] The influence of the PBP

(pentagonal-bipyramidal) geometry has an important impact on the physical and

chemical properties of this class of complexes. In the PBP geometry with pentadentate

ligands in equatorial plane, two monodentate ligands in the apical positions are

relatively loosely bound to the metal center, making such structures reactive in terms

of substitution processes.[3] During the catalytic SOD cycle a solvent molecule in

those apical positions can be easily substituted by superoxide in an inner sphere redox

mechanism. It is still questionable whether this substitution process on seven

coordinate manganese complexes has more dissociative or associative character. In the

literature in has been postulated that the water dissociation and formation of six

coordinate intermediate is a rate determining step in the inner sphere pathway of SOD

cycle.[4] At the same time the studies in our group on water exchange processes on the

proven seven coordinate SOD mimetics[5] have demonstrated that this can not be the

case, since the second order rate constants kIS for the inner-sphere catalytic pathway are

significantly higher than the corresponding kex/[H2O] values for the water-exchange.

The mechanism of the water exchange on the seven-coordinate MnII center has an

interchange (Id) character, where the incoming water molecule also plays a role in the

overall substitution process.[6] In the case of a negatively charged nucleophile it is to

be expected that a substitution mechanism gains more an associative character.

Interestingly, our seven-coordinate FeIII SOD mimetics can undergo substitution

reactions within an associative interchange (Ia) mechanism, suggesting that in a

transition state a sort of a eight-coordinate structure exists. Therefore we wanted to

demonstrate existence of an eight-coordinate MnII species by synthesizing and

characterizing such a complex. At the same time, by studying the reaction of a

coordination saturated and consequently a substitution inert manganese species with


81

superoxide, we can further probe whether the SOD catalysis can be achieved by an

outer sphere mechanism, which has been proposed as a parallel pathway operating

within the seven-coordinate MnII SOD mimetics.[7]

To this goal in the present chapter we report a synthesise of a new

eight-coordinate manganese complex [MnII(Hdapmp)2](ClO4)2(H2O)2 (7) (Hdapmp

= [1-(6-acetyl-2-pyridinyl) ethylidene] hydrazone ), and we studied its reactivity

toward superoxide by electrochemical, spectrophotometrical and submillisecond

mixing UV/vis stopped-flow measurements.

Regarding structural features that might have an influence on a complex

reactivity toward superoxide we synthesizes and characterized a seven-coordinate

μ-oxo-dimer iron(III) [(H2O)2FeIII2(Daphp)2O](ClO4)4(H2O)3 (8) complex. It has

been observed in the literature that as the concentration of Fe or Mn SOD catalysts

increases, the catalyst loses activity. Namely, the rate does not increase in a linear

fashion as the catalyst concentration increases. This phenomenon has been explained by

formation of oxo- or hydroxo-bridged dimmers. Those dimers may not possess the SOD

catalytic activity, and as the concentration of complex increases, the dimer formation

reaction becomes favored, thereby lowering the apparent catalytic activity.[7] In order to

prove such a paradigm, herein we have also tested the reactivity of our μ-oxo-dimer

iron(III) complex toward superoxide.


For Materials and Instrumentation and Measurements see Chapter 2.

Synthesis of Hdapmp ligand[8]

A solution of 2,6-diacetylpyridine (8.15 g, 50mmol) in absolute ethanol (150mL) was

added dropwise with stirring a solution of 2-Hydrazinopyridine (5.45 g, 50mmol)

dissolved in the same solvent, and the mixture left to react under nitrogen for 24 hours.

The resulting pale yellow solution was concentrated on a rotary evaporator until a


82

white product began to precipitate. After being kept in the refrigerator for one hour, the

precipitate product was filtered and recrystallized from acetone, which was identified

as pure Daphp ligand. The filtered solution was evaporated to dryness and the crude

solid residue was dissolved in 5mL acetate- hexane (70 : 30) solvent, which was then

adsorbed on a silica column (80 x 3 cm) pretreated with ethyl acetate -hexane (70 : 30)

and eluted with the same mixture of solvents. Thus the light yellow produce Hdapmp

(2.6 g, yield 20.3%) was separated and identified analytic purity. Anal. calcd. for

C14H16N4O: C, 65.61, H, 6.29, N, 21.86%. Found: C, 65.38, H, 5.87, N 21.71%.

Synthesis of [MnII(Hdapmp)2](ClO4)2(H2O)2 (7)

To a suspension of Hdapmp ligand (0.256g 1mmol) in 40mL methanol was dropwise

added a 20mL methanol solution of Mn(ClO4)2·4H2O (0.326 g, 1 mmol). The mixture

was refluxed for two hours with stirring and filtered. The pale yellow filtered solution

was concentrated on a rotary evaporator until a pate yellow product began to

precipitate. After being kept in the refrigerator for one hour, the precipitate product

was filtered and recrystallized in acetonitrile-methanol (1:1), rendering yellow

crystals of the compound suitable for X-ray diractometry. IR data (KBr, cm–1):

3352s(NH), 3233s(H2O), 3129s, 2335m, 1672s(C=O), 1522m, 1345m(amide), 1273m,

1240m(CH) 1154w, 1076m, 800w, 779m, 636m(py). Anal. calcd. for

C28H36N8O10Cl2Mn: C, 43.65, H, 4.71, N, 14.54%. Found: C, 44.02, H, 4.87, N 14.31%.

Synthesis of [(H2O)FeIII(Daphp)OFeIII(Daphp)(H2O)](ClO4)4(H2O)3 (8)

2,6-diacetylpyridine (1.63g, 10 mmol) and 2-Hydrazinopyridine (2.18 g, 20 mmol)

were added to 60 mL of methanol and the mixture was stirred at 55oC for one hour.

Fe(ClO4)3·6H2O (4.26 g, 10 mmol) dissolved in 20mL methanol was dropwise added

into the resulting white suspension. The solution color changed to red and resulted in a

clear dark red solution. After 3 hours reflux the hot reaction mixture was filtered

and the dark residue discarded. The dark red solution was then allowed to cool to

room temperature and kept in refrigerator, rendering dark red crystals of the

compound, suitable for X-ray diractometry.(yield: 3.67 g, 57.8%).. IR data (KBr,


83

cm–1): 3335s(NH), 3241s(H2O), 3119s, 2337m, 1692s, 1537m, 1341m(amide), 1284m,

1241m(CH) 1117w, 1085m, 804m, 777m, 632m(py). Anal. calcd. for

C38H52Cl4N14O24Fe2: C, 34.00, H, 3.90, N, 14.61%. Found: C, 34.32, H, 3.87, N

14.31%.

X-ray Crystal Structure Determinations

Data for 7 and 8 were collected at 100 K using a Bruker-Nonius KappaCCD

diffractometer (λ = 0.71073 Å, graphite monochromator). Data were measured using ω

scans of 0.3o per frame for 30 s, such that a hemisphere was collected. The first 50

frames were re-collected at the end of data collection to monitor for decay. All data

sets were corrected for Lorentz and polarization effects. Absorption effects were taken

into account by semiempirical methods using either multiple scans (SADABS).[9] The

structures were solved by direct methods and refined using full-matrix least-squares

procedures on F2 (SHELXTL NT 6.12).[10] The positions of all hydrogen atoms were

derived from difference fourier maps. The isotropic displacement parameters of all

hydrogen atoms were tied to those of the equivalent isotropic displacement parameters

of their corresponding C, N or O carrier atoms. Crystal data, data collection parameters

and refinement details of the structure determinations of complexes 7 and 8 are

summarized in Table 4-1


84

DFT calculation:

The structure was pre-optimized with UHF/LANL2MB[MB1,MB2,LANL2DZp-2]

and characterized as a minimum by computation of vibrational frequencies.[11-14]

The B3LYP hybrid density functional [B3LYP-1, B3LYP-2, B3LYP-3] and the

LANL2DZ basis set augmented with polarization functions (further denoted as

LANL2DZp) [LANL2DZp-1, LANL2DZp-2, LANL2DZp-3, LANL2DZp-4] was used

Complex 7 8

Empirical formula C28H34Cl2N8O13Mn C38H52Cl4N14O24Fe2

Formula weight 816.47 1342.44

Temperature (K) 100(2) 100(2)

Wavelength (Å) 0.71073 0.71073

Crystal system Triclinic Monoclinic

space group P1 P2(1)/c

a (Å) 8.9793(8) 14.391(2)

b (Å) 9.2906(2) 28.470(2)

c (Å) 10.9279(8) 14.745(2)

α(°) 104.375(3) 90

β (°) 94.750(6) 117.99(1)

γ (°) 91.618(4) 90

V (Å3) 878.9(1) 5334.6(9)

Z 1 4

ρ (Mg/m3) 1.543 1.671

Absorption coefficient 0.603 0.841

F(000) 421 2760

Crystal size (mm) 0.32 x 0.23 x 0.10 0.14 x 0.14 x 0.13

Theta range for data

collection.

3.29 to 27.88 3.11 to 27.10

Limiting indices -11<=h<=11, -12<=k<=12,

-14<=l<=14

-18<=h<=18, -36<=k<=36,

-18<=l<=18

Reflections collected /

unique

22840 / 8179 [Rint = 0.0548] 63163 / 11753 [Rint = 0.0507]

Max. and min. transmission 0.941 and 0.849 1.000 and 0.937

Data/restraints / parameters 8179 / 5 / 600 11753 / 294 / 957

GooF 0.889 1.041

R1 0.0442 0.0476

R2 (all data) 0.0887 0.1167

Largest diff. peak (e Å-3) 0.341 and -0.409 0.930 and -0.754

Table 4-1 Crystal data and structure refinement for 7-8


85

for structure refinement, additional we tested the stability of the wave function.[15]

The Gaussian 03 suite of programs was used [G03]. Further structure optimizations

were carried out with Jaguar 6.5 [Jag] applying the pure density functional BP86

[BP86] together with the LACVP* basis set [LANL2DZp-2,BASIS] for structure

calculations in the gas phase [BP86/LACVP*] and the optimization including the

water model BP86(H2O)/LACVP* [JAG-SOLV] as implemented in Jaguar 6.5.[16,

17]


4.4.1 Studies on the complex 7

Structure:

The complex 7 crystallizes in the triclinic space group P1, which is one of the acentric

space groups. The acentric unit consists of one cationic complex, two perchlorate

anions and two lattice water molecules. The X-ray structure of this complex shows that

the manganese atom is coordinated by six nitrogen atoms and two oxygen atoms from

a b Figure 4-1 a) ORTEP view of [MnII(Hdapmp)2]2+ in the crystal of 7 drawn with thermal elliposide at 50% probability level, perchrorate anions are omitted for clarity, b) a view of the coordination environment and the coordination polyhedron


86

the two Hdapmp ligands and gives a N6O2 eight-coordinate environment. (Figure 4-1)

The main bond lengths and angles are listed in Table 4-2. Comparison of the obtained

structural parameters with the corresponding values for the ideal eight-coordinate

geometry[18] shows that the geometry of 7 is closer to a distorted-dodecahedron than

to an antiprism. A view of the coordination environment and the coordination

polyhedron is shown in Figure 4-1b. The main cause of distortion is the asymmetric

Hdapmp ligand, which offers three N and one O donor atoms causing a difference

between the Mn-N and Mn-O bond distances. Another factor that contributes to the

distortion is the difference between the chelate modes of two Hdapmp ligands. The

Mn1 center is 0.08 Å out of the best equatorial plane which is defined by Mn1, N1, N2,

N4, and O1 (deviations of other atoms from the mean plane are +0.0253, -0.0022,

+0.018, and +0.0388 Å respectively, which give a mean deviation from plane about

0.0328 Å ). Mn1–N bond lengths range from 2.334(3) to 2.346(2) Å, which gives an

average Mn1–N bond length of 2.339 Å, whereas the Mn1–O bond length is 2.497(2)

Å. Adjacent N–Mn1–N(O) bond angles range from 66.21(8) to 68.87(9)o, with the

disjunctive N4-Mn1-O1 angle of 156.64(8)o. In the second coordinated Hdapmp

ligand, Mn1 is 0.027 Å out of the best equatorial plane which is defined by Mn1, N5,

N6, N8, and O2 and its mean deviation from plane is about 0.0235 Å. Mn1–N bond

lengths with this ligand range from 2.316(3) to 2.340(3) Å, which gives an average

Mn1–N bond length of 2.328 Å and Mn1–O is 2.441(2) Å. The average Mn1–N and

Mn1–O bond length in this ligand are a little bit shorter than those in the case of the

firs Hdapmp ligand. The two Hdapmp ligands are in almost ideal orthogonal

arrangement, with the dihedral angle between them being 90.8o.


87

In the crystal packing of [MnII(Hdapmp)2]2+, all four pyridine rings are involved in

π−π stacking interactions with adjacent complex cations. As shown in Figure 4-2a,

each pair of adjacent MnII atoms are related via this aromatic π−π stacking

interactions to form a zigzag chain running along a-direction. The face-to-face distance

between the paired pyridine rings is ca. 3.364 Å and the dihedral angle between them

is just 2.4O, which indicates that there is a strong aromatic π−π stacking interaction.[19,

20, 21] The adjunct Mn…Mn distance in this chain is 8.979 Å. It is worth noting that

the lateral pyridine rings from adjacent chains are further paired to furnish another kind

Bond Distances (Å) Mn(1)-N(5) 2.316(3) O(1)-C(12) 1.219(4)

Mn(1)-N(8) 2.317(2) O(2)-C(26) 1.214(4)

Mn(1)-N(2) 2.334(3) N(2)-C(6) 1.299(4)

Mn(1)-N(4) 2.338(3) N(2)-N(3) 1.361(4)

Mn(1)-N(6) 2.340(3) N(6)-C(20) 1.288(4)

Mn(1)-N(1) 2.346(2) N(6)-N(7) 1.363(4)

Mn(1)-O(2) 2.441(2)

Mn(1)-O(1) 2.497(2)

Bond Angles (deg) N(5)-Mn(1)-N(8) 136.56(9) N(2)-Mn(1)-N(1) 67.48(9)

N(5)-Mn(1)-N(2) 130.43(9) N(4)-Mn(1)-N(1) 136.16(9)

N(8)-Mn(1)-N(2) 87.46(9) N(6)-Mn(1)-N(1) 130.73(9)

N(5)-Mn(1)-N(4) 90.12(9) N(5)-Mn(1)-O(2) 67.28(8)

N(8)-Mn(1)-N(4) 84.93(9) N(8)-Mn(1)-O(2) 156.00(8)

N(2)-Mn(1)-N(4) 68.87(9) N(2)-Mn(1)-O(2) 69.82(8)

N(5)-Mn(1)-N(6) 67.54(9) N(4)-Mn(1)-O(2) 93.58(9)

N(8)-Mn(1)-N(6) 69.12(9) N(6)-Mn(1)-O(2) 134.81(8)

N(2)-Mn(1)-N(6) 148.05(9) N(1)-Mn(1)-O(2) 75.23(8)

N(4)-Mn(1)-N(6) 87.09(9) N(5)-Mn(1)-O(1) 78.42(8)

N(5)-Mn(1)-N(1) 121.72(9) N(8)-Mn(1)-O(1) 89.52(8)

N(8)-Mn(1)-N(1) 89.28(8) N(2)-Mn(1)-O(1) 133.62(8)

N(1)-Mn(1)-O(1) 66.21(8) N(4)-Mn(1)-O(1) 156.64(8)

O(2)-Mn(1)-O(1) 100.45(8) N(6)-Mn(1)-O(1) 69.77(8)

Table 4-2 Selected bond lengths (Å) and bond angles (deg) of complex 7


88

of π−π aromatic stacking interactions, which is also in an offset fashion with a

face-to-face distance of ca. 3.504 Å and extend the zigzag chains into wavy 2-D layers

parallel to the ab plane, as illustrated in Figure 4-2b.

Calculated eight-coordinate structure of 7

In the solid state structure the geometry of the complex cation is usually

influenced by the packing effects and different intra- and intermolecular secondary

interactions. Therefore we have performed the DFT calculations, in order to obtain the

structural parameters that are not affected by secondary interactions. In a gas-phase the

structure of 7 has a C2 symmetry. Independent from the selected DFT-method and

whether solvent effects have been included into the calculations or not, all calculated

Mn-N bonds are a little bit longer and the calculated Mn-O bonds are somewhat

shorter than those in the solid state structure (Table 4-3). The obtained Mn-N bond

distances (2.35–2.40 Å) are in the range expected for the coordination of

sp2-hybridized nitrogen to MnII center. To the best of our knowledge this compound

a b Figure 4-2 a) Chain formed with [MnII(Hdapmp)2] molecule squares via π−π interaction along a direction b) Further view of 2D structure along the c direction formed with [MnII(Hdapmp)2]2+ molecule squares via two kind of π−π interactions


89

shows for the first time an interaction between the manganese center and the sp2-

hybridized oxygen atom (2.4 Å), which is not a part of an amide functional group,

within an eight-coordinated geometry. For the comparison, the usual Mn-Oamide bond

length in the published eight-coordinate structures is 2.41 Å.[22]

Redox Propertries of 7

0.2 0.4 0.6 0.8 1.0 1.2 1.4

-1.5x10-5

-1.0x10-5

-5.0x10-6

0.0

5.0x10-6

1.0x10-5

i / A

E / V vs Ag/AgCl

Wavelength (nm)

Abs

orba

nce

325 375 425 475 525 575 625 675

0,10

0,30

0,50

0,70

0,90

1,10

1,30

1,50

1,70

Time (sec)

Abs

orba

nce

2,5 7,5 12,5 17,5 22,5

0,90

1,10

1,30

1,50

1,70452,4 (nm)

Wavelength (nm)

Abs

orba

nce

325 375 425 475 525 575 625 675

0,10

0,30

0,50

0,70

0,90

1,10

1,30

1,50

1,70

Time (sec)

Abs

orba

nce

2,5 7,5 12,5 17,5 22,5

0,90

1,10

1,30

1,50

1,70452,4 (nm)

a b Figure 4-4 a) Cyclic voltammogramms of 7 purged with nitrogen. Conditions: [7] = 0.5 x 10-3 M, [Bu4NBF4] = 0.1 M, T = 298 K, scan rates = 0.1 V/s. b) Time resolved UV/vis spectra recorded for the reaction of 7 (1 x 10-4 M) with 1 mM KO2 in DMSO at room temperature at time intervals of 0.03 s (total observation time 27 s

[Å] x-ray B3LYP/LANL2DZp BP86/LACVP* BP86(H2O)/LACVP*

P.G. C1 C2 C2 C2

Mn-N1 2.346/2.316 2,39 2,35 2,36

Mn-N2 2.334/2.340 2,40 2,38 2,38

Mn-N4 2.338/2.317 2,38 2,38 2,38

Mn-O1 2.497/2.441 2,41 2,40 2,43

Table 4-3 Calculated structural data


90

The cyclic voltamogram of complex 7 in DMSO purged with nitrogen is given

in Figure 4-4a. When scan moves from 1.5 V to 0 V at a 100 mV s-1 scan rate, one

cathodic peak appears at 1.221 V vs Ag/AgCl, but it has no corresponding anodic wave

in this range. 7 also shows one reduction process at Epc = 581 mV followed by the

corresponding oxidation potential at 820 mV vs Ag/AgCl (obtained by calibration with

ferrocene), which is due to the MnIII/MnII redox couple. The anodic to cathodic peak

separation is quite large (239 mV at 100 mV s-1) and increases with scan rates,

indicating that the oxidation of 7 is a quasireversible process. It should be mentioned

that in comparison to the manganese complex with the analogue bis(hydrazone) ligand,

[MnII(Daphp)(H2O)2](ClO4)2, the [MnII(Hdapmp)2]2+ complex containing two

monohydrazones of diacetyl pyridine has a somewhat lower redox potential,

suggesting that the presence of four coordinated pyridine groups have an certain

stabilizing effect on MnIII form of the complex. We can only speculate that the

oxidation of 7 is followed by breaking of the two Mn-O bonds and formation of

six-coordinate MnIII species, with two Hdapmp acting as a three-dentate ligands, since

the much smaller Mn3+ cation with the d4 configuration can hardly accommodate the

eight-coordinate geometry. The redox potential of [MnII(Hdapmp)2]2+ is similar to the

redox potentials of SOD active [MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2 and the

proven macrocyclic SOD mimetic [MnII(Me2[15]pyridinaneN5)(H2O)2]Cl2 in DMSO

(see Chapter 2).

Reaction with Superoxide

When the excess of KO2 (1 mM) is mixed with a solution of 7 (1 x 10-4 M)in

DMSO containing a controlled amount of water (0.06%), only a small spectral change

at 270 nm is observed, accompanied by the prominent absorbance increase in the range

of 360-650 nm (Figure 4-4b). The maximum absorbance change in that spectral range

is achieved in about 30 s and is followed by a slow decrease of the same absorption

band. The small absorbance change at 270 nm (the wavelength characteristic for

superoxide) clearly demonstrates that in the presence of 7 there is no catalytic

decomposition of superoxide. The small decrease of a band at 270 nm is caused by


91

spontaneous superoxide decomposition due to the presence of 0.06 % of water in

DMSO.[23] The absorption increase in 360-650 nm range is most probably due to the

stoichiometic oxidation of manganese center. (Figure 4-4b) The obtained MnIII species

is not very stable and either a slow dechelation may occur or its reduction by H2O2,

which is generated in solution as a product of spontaneous superoxide decomposition.

Since the coordination sphere around the manganese center of 7 is already saturated by

eight donor atoms, the oxidation of 7 by superoxide most probably follows an outer

sphere electron transfer mechanism. These results demonstrate that the substitution

inert complexes can not be considered as SOD mimetics.

4.4.2 Studies on the complex 8

Sturcture

The perchlorate salt of the FeIII complex of Daphp ligand, 8, crystallizes in the

monoclinic space group P21/c (see Table 4-1 for pertinent crystal data). A view of the

unit of this complex with main atom numbering scheme is shown in Figure 4-5.

Selected bond lengths and angles are summarised in Table 4-4. The bridging oxo atom

Figure 4-5 ORTEP view of [(H2O)FeIII(Daphp)(O)FeIII(Daphp)(H2O)]4＋ in the crystal of 7 drawn with thermal elliposide at 65% probability level and main atom numbering scheme , four perchrorate ions are omitted for clarity.


92

O1 connects two [FeIII(Daphp)(H2O)] monomers and forms a cationic dimer (the

Fe1…Fe2 distance 3.569Å and Fe1–O1–Fe2 167.08(14)O).

Both of these two Fe atoms are in the distorted pentagonal-bipyramidal coordination

sphere with five nitrogen donor atoms from chelating Daphp ligand in the equatorial

Bond Distances (Å) Fe(1)-O(1) 1.803(2) Fe(2)-O(1) 1.789(2)

Fe(1)-O(2) 2.109(2) Fe(2)-O(3) 2.149(2)

Fe(1)-N(1) 2.213(3) Fe(2)-N(8) 2.232(3)

Fe(1)-N(4) 2.227(3) Fe(2)-N(14) 2.246(3)

Fe(1)-N(7) 2.237(3) Fe(2)-N(9) 2.249(3)

Fe(1)-N(5) 2.246(3) Fe(2)-N(11) 2.250(3)

Fe(1)-N(2) 2.248(3) Fe(2)-N(12) 2.257(3)

N(2)-C(6) 1.289(4) N(9)-C(25) 1.302(4)

N(2)-N(3) 1.359(4) N(9)-N(10) 1.350(4)

N(5)-C(13) 1.284(4) N(12)-C(32) 1.288(4)

N(5)-N(6) 1.364(4) N(12)-N(13) 1.356(4)

Bond Angles (deg)

Fe(2)-O(1)-Fe(1) 167.08(14)

O(1)-Fe(1)-O(2) 177.72(10) O(1)-Fe(2)-O(3) 178.32(10)

O(1)-Fe(1)-N(1) 93.32(10) O(1)-Fe(2)-N(8) 96.33(9)

O(2)-Fe(1)-N(1) 87.37(10) O(3)-Fe(2)-N(8) 85.01(9)

O(1)-Fe(1)-N(4) 100.71(10) O(1)-Fe(2)-N(14) 102.21(10)

O(2)-Fe(1)-N(4) 77.33(10) O(3)-Fe(2)-N(14) 77.56(10)

N(1)-Fe(1)-N(4) 135.82(10) N(8)-Fe(2)-N(14) 131.74(10)

O(1)-Fe(1)-N(7) 86.41(9) O(1)-Fe(2)-N(9) 100.85(10)

O(2)-Fe(1)-N(7) 94.54(10) O(3)-Fe(2)-N(9) 78.65(10)

N(1)-Fe(1)-N(7) 138.07(10) N(8)-Fe(2)-N(9) 69.41(9)

N(4)-Fe(1)-N(7) 84.89(10) N(14)-Fe(2)-N(9) 145.85(9)

O(1)-Fe(1)-N(5) 104.85(9) O(1)-Fe(2)-N(11) 85.86(9)

O(2)-Fe(1)-N(5) 77.43(10) O(3)-Fe(2)-N(11) 92.46(9)

N(1)-Fe(1)-N(5) 69.56(10) N(8)-Fe(2)-N(11) 140.33(10)

N(4)-Fe(1)-N(5) 142.54(9) N(14)-Fe(2)-N(11) 85.62(9)

N(7)-Fe(1)-N(5) 70.05(10) N(9)-Fe(2)-N(11) 71.28(9)

O(1)-Fe(1)-N(2) 84.46(9) O(1)-Fe(2)-N(12) 84.89(10)

O(2)-Fe(1)-N(2) 93.75(10) O(3)-Fe(2)-N(12) 96.56(10)

N(1)-Fe(1)-N(2) 69.60(10) N(8)-Fe(2)-N(12) 68.73(9)

N(4)-Fe(1)-N(2) 70.36(10) N(14)-Fe(2)-N(12) 69.08(9)

N(7)-Fe(1)-N(2) 151.42(10) N(9)-Fe(2)-N(12) 138.12(10)

N(5)-Fe(1)-N(2) 138.52(10) N(11)-Fe(2)-N(12) 150.44(9)



93

plane and one oxygen atom from the coordinated water molecule and the bridging

oxygen atom (O1) in the axial positions. Fe1 is 0.126 Å out of the best equatorial plane

which is defined by Fe1, N1, N2, N4, N5 and N7 (deviations of N atoms from the

mean plane are +0.012, +0.2104, +0.0102, -0.3114 and +0.355 Å respectively, which

gives a mean deviation from plane of about 0.234 Å ). Fe1–N bond lengths range from

2.213(3) to 2.248(3) Å, which gives an average Fe1–Npyridine bond length of 2.226 Å

and an average Fe1–Nimine length of 2.247 Å. The Fe1–O and Fe1–Ooxo distances are

2.109(2) Å and 1.803(2) Å respectively. The adjacent N–Fe1–N bond angles range

from 69.56(10) to 70.36(10)o, and are slightly smaller than the ideal value of 72O for a

pentagonal-bipyramidal arrangement. The disjunctive N4-Fe1-N7 angle is 84.89(10)o

and O1-Fe1-O2 angle is 177.72(10)o. The dihedral angle between the five-member ring

[N2–N3–C8–N4–Fe1] and disjunctive [N5–N6–C15–N7–Fe1] ring from Daphp

ligand is 29.6O, which indicates the helical conformation of the pentadentate ligand

around the metal center. Compared to the coordination environment of Fe1, the

coordination environment of Fe2 center is very similar. Fe2 is 0.129 Å out of the best

equatorial plane, which is defined by Fe2, N8, N9, N11, N12 and N14 (deviations of N

atoms from the mean plane are +0.082, +0.225, -0.305, -0.286 and +0.412 Å

respectively, which give a mean deviation from plane about 0.240 Å). The Fe2–O

distance is 2.149(2) Å and the Fe2–Ooxo is 1.789(2) Å. Fe2–N bond lengths range from

2.232(3) to 2.257(3) Å, giving an average Fe2–Npyridine bond length of 2.243 Å and an

average Fe2–Nimine bond length of 2.253 Å. These bonds are a bit longer than the

corresponding bond lengths of Fe1, which is caused by the somewhat shorter Fe2–Ooxo

bond resulting in the bigger deviation of Fe2 center from an ideal

pentagonal-bipyramidal geometry. The adjacent N–Fe2–N bond angles range from

68.73(9) to 71.28(9)o. The disjunctive N11-Fe2-N14 angle is 85.62(9)o and O1-Fe2-O3

angle is 178.32(10)o. The dihedral angle between the five-member ring

[N9–N10–C27–N11–Fe2] and disjunctive [N12–N13–C34–N14–Fe2] ring is 27.1O,

demonstrating the helical conformation of the pentadentate ligand around the Fe2 atom

as well.


94

Electrochemical behaviour and reaction with superoxide

The electrochemical measurements of the iron(III) μ-oxo dimer complex 8

suggests that the dimer structure is stable in DMSO. When scan proceeds from 1 V

towards -0.8 V and then turns back to 1 V, reduction peaks occur at 427, 248, -302 and

-566 mV and oxidations are seen at -386, 323 and 491 mV (Figure 4-6). The two

reduction peaks in the negative potential range correspond to the redaction of two iron

centers. The quite negative potentials are the consequence of the oxo bridging ligand,

which strongly stabilizes the Fe3+ oxidation state. After reduction, the dimer structure

does not exist any more and therefore in the reverse scan only one oxidation peak has

been observed, which corresponds to a monomeric species. The existence of two peaks

in the positive potential range has already been reported for some iron μ-oxo

dimers.[24] It can be postulated that the presence of five nitrogen atoms, three of them

from the pyridine groups, stabilizes higher oxidation states of two iron centers.

Consequently, these two peaks can be assigned to the Fe(IV)/Fe(III) redox couples.

Both of these two waves have a 60-70 mV separation between Epa and Epc, indicating

reversible electrode reactions which involve transfer of one electron per dimeric unit,

further suggesting a presence of two non equivalent redox active iron centers.

Reversibility of these two processes and relatively low potentials that they require

-1.0 -0.5 0.0 0.5 1.0-6.0x10-5

-4.0x10-5

-2.0x10-5

0.0

2.0x10-5

4.0x10-5

i, A

E(V) vs Ag/AgCl

Figure 4-6. Cyclic voltammogramms of 8 purged with nitrogen Conditions: [8] = 0.5 x 10-3 M, [Bu4N·BF4] = 0.1 M, T = 298 K, scan rates = 0.2 V/s.


95

makes further detailed investigations of the redox behavior of our iron(III) μ-oxo

dimer highly attractive.

The rapid-scan UV-vis spectral measurements of the reaction between the dimer

complex and superoxide in DMSO solution containing 0.06% of water (Figure 4-7a)

demonstrate that the dimer is not able to catalytically decompose superoxide. Namely

the expected fast and prominent decrease of the superoxide absorption band with the

maximum at around 270 nm is not observed. However, a buildup of an intense

absorption band in the 375-550 nm region is observed. The preliminary kinetic

measurements (a detailed study is out of the scope of the present work) of the reaction

between 1 mM superoxide and 2 x 10-5 M complex have revealed that the reaction

proceeds in a two consecutive steps. The corresponding kinetic trace (Figure 4-7b) can

be best fitted with a double exponential function. The spectral changes related to these

two processes (Figure 4-7b) exhibit the same character. These results suggest that

superoxide reacts with two chemically similar metal centers. At the present stage we

can only postulate that the labile solvent molecules, coordinated to the each iron center

in the apical positions trans to the oxo bridge, can be substituted by two superoxide

Wavelength (nm)

Abs

orba

nce

350 400 450 500 550 600 6500,0

0,20

0,40

0,60

0,80

1,00

Time (sec)A

bsor

banc

e0,050 0,150 0,250 0,3500,4500,5500,6500,7500,850

0,250

0,350

0,450

0,550

0,650

0,750 454,9 (nm)

a b

Figure 4-7. a) Time resolved UV/vis spectra recorded for the reaction of 8 (2 x 10-5 M) with 1 mM KO2 in DMSO at room temperature at time intervals of 3 ms (total observation time 1.08 s). b) kinetic traces of reaction at 455 nm in 1.08 s


96

anions. Whether this substitution process is followed by electron transfer and/or

decomposition of the dimer structure remains to be seen.

4.5 Conclusion

Because of the lack of ligand field stabilization energy for the high-spin d5

configuration, ligand steric and electronic effects appear to play a major role in

defining the coordination number and geometry in high-spin MnII complexes.[2]

Herein we have synthesized and characterized an eight coordinate high-spin MnII

complex 7 of Hdapmp ligand, which is a monohydrazide of 2,6-diacetylpyridine. Two

such tetradentate ligands are coordinated to the MnII center, and are in the orthogonal

position to each other. Although the redox potential of the MnIII/MnII couple of 7 is

similar to the redox potentials of some proven SOD mimetics,[25] the studied

eight-coordinated MnII complex demonstrate no ability for catalytic decomposition of

superoxide. This can be explained in terms of the saturated coordination geometry

around the metal center, and shows that for the SOD activity, the complex redox

potential is not the only important requirement. The efficient catalysis seems to be

facilitated by binding of superoxide to the redox-active metal center within an

inner-sphere electron transfer mechanism.

Regarding the iron μ-oxo dimers, our experimental studies confirmed for the

first time the literature postulation that μ-oxo dimer structures do not posses SOD

catalytic activity. However, we could demonstrate that a stoichiometric two step

reaction between the FeIII μ-oxo dimer and superoxide is possible. This reaction most

probably involves a substitution of the labile solvent molecules at two iron center s,

which are in the trans position to the μ-oxo group, by two superoxide anions. Detailed

nature and the mechanism of the observed process will be a subject of some further

studies.

4.6 References


97

[1] M. Louloudi, V. Nastopoulos, S. Gourbatsis, S. P. Perlepes, N. Hadjiliadis,

Inorg. Chem. Comm., 1999, 2, 479–483.

[2] M. Mikuriya, Y. Hatano, E. Asato, Bull. Chem. Soc. Jpn., 1997, 70, 2495.

[3] I. Ivanovic-Burmazovic, A. Bacchi, G. Pelizzi, V.M. Leovac, K. Andjelkovic,

Polyhedron, 1998, 18, 119.

[4] D. P. Riley, S. L. Henke, P. J. Lennon, and K. Aston, Inorg. Chem. 1999, 38,

1908-1917.

[5] A. Dees, A. Zahl, R. Puchta, N. J. R. van Eikema Hommes, F. W. Heinemann,

I. Ivanovic-Burmazovic, Inorg. Chem. 2007, 46, 2459

[6] H. H. Loffler, A. M. Mohammed, Y. Inada, S. Funahashi, J. Comput. Chem.,

2006, 27(16), 1944-1949.

[7] D. P. Riley, Chem. Rev. 1999, 99, 2573-2587.

[8] M. Giampaolo, P. Gino, J. Chem. Soc., Dalton Trans., 1981, 2, 357-361

[9] SADABS, 2.06, Bruker-AXS, Inc., 2002, Madison, WI, U.S.A.

[10] SHELXTL NT 6.12, Bruker-AXS, Inc., 2002, Madison, WI, U.S.A.

[11] P. J. Hay, W. R. Wadt, J. Chem. Phys., 1985, 82, 299-310.

[12] P. J. Stephens, F. J. Devlin, C. F. Chabalowski, M. J. Frisch, J. Phys. Chem.,

1994, 98, 11623-11627.

[13] P. J. Hay, W. R. Wadt, J. Chem. Phys., 1985, 82, 270-283

[14] S. Huzinaga (Ed.), Gaussian Basis Sets for Molecular Calculations, Elsevier,

Amsterdam 1984.

[15] S. Klaus, H. Neumann, H. Jiao, A. Jacobi von Wangelin, D. Gördes, D.

Strübing, S. Hübner, M. Hately, C. Weckbecker, K. Huthmacher, T. Riermeier,

M. Beller, J. Organomet. Chem., 2004, 689, 3685-3700.

[16] R. Puchta, R. van Eldik Eur. J. Inorg. Chem., 2007, 1120 - 1127.

[17] R. Puchta, R. Meier, R. van Eldik, Aust. J. Chem., 2007, 60, 889–897. and

literature cited therein.

[18] D. Casanova, M. Llunell, P. Alemany and S. Alvarez, Chem. Eur. J. 2005, 11,

1479–1494.

[19] B. Moulton, M. J. Zaworotko，Chem. Rev. 2001, 101, 1629.


98

[20] G.-F. Liu, B.-H. Ye, Y.-H. Ling, X.-M. Chen, Chem. Comm., 2002, 14,

1442-1443.

[21] X. M. Chen, G. F. Liu, Chem. Eur. J., 2002, 8, 4811–4817.

[22] M. Louloudi, V. Nastopoulos, S. Gourbatsis, S. P. Perlepes, N. Hadjiliadis,

Inorg. Chem. Comm., 1999, 2, 479–483.

[23] D.-H. Chin, G., Jr. Chiericato, E. J., Jr. Nanni, D. T. Sawyer, J. Am. Chem.

Soc. 1982, 104, 1296-1299.

[24] T. Glaser, R. H. Pawelke, M. Heidemeier, Z. Anorg. Allg. Chem. 2003, 629,

2274-2281.

[25] S. Durot, C. Policar, F. Cisnetti, F. Lambert, J.-P. Renault, G. Pelosi, G. Blain,

H. Korri-Youssoufi and J.-P. Mahy, Eur. J. Inorg. Chem. 2005, 3513-3523

Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation

99

Chapter 5

5.1 Abstract

In this chapter we report that a family of dendrimeric fullerene derivatives

II-VII, with a variety of attached dendrimeric groups, has been synthesized and

evaluated as superoxidise dismutase mimetics. As evidenced by electrochemical,

spectrophotometrical and sub-millisecond mixing UV/vis stopped-flow measurements,

as well as by indirect SOD assays and H2O2 detection, carried out in DMSO solutions

or in aqueous solution at pH = 7.8, some of these species (II-V) show that they are

capable of removing the superoxide radical with catalytic rate constants, which is very

similar to the results obtained from the indirect cytochrome c assay. Importantly, this is

the first time that the catalytic SOD activity of fullerenes has been detected by using a

direct stopped-flow method, where a high excess of superoxide over fullerene can be

utilized, and there is no interference with additional components in the solution.

However, VI and VII do not induce catalytic decomposition of O2·—, which

additionally implies that the architecture of fullerene derivatives affects their reactivity

toward O2·— and increases further our knowledge about the structure–activity

relationship of C60 derivatives as superoxide dismutase mimetics. By studying this

series of fullerenes, for the first time we could observe that there is a direct correlation

between redox and structural properties of the studied fullerenes and their SOD

High Catalytic Activity of Dendritic C60 Monoadducts in

Metal-Free Superoxide Dismutation


100

activity, namely, the higher the redox potential (ability to be reduced by O2·—) the

higher the SOD activity.

5.2 Introduction

Water soluble fullerenes, in particular the tris-malonyl-C60 derivative I (so

called C3)[1] have been shown to exhibit strong anti-oxidant activity against reactive

oxygen species (ROS) in vitro and to protect cells and tissue from oxidative injury and

cell death in vivo.[2] Especially the ability to destroy the toxic superoxide O2·— was

suggested to be responsible for fullerene anti-oxidant activity,[3] although its

mechanism is still not clear. Dugan and coworkers offered evidence in support of

catalytic superoxide dismutation mechanism instead of direct radical attack on the C60

moiety of I showing that it could act as a metal free mitochondrial manganese

superoxide dismutase (MnSOD) mimetic.[3a] They proposed a complex formation

between C3 and O2·—. In this work we present for the first time clear and unambiguous

evidence for a catalytic dismutation process whose key steps are successive O2·—

oxidation, within an outer sphere electron transfer process, and fullerene derivative

mediated O2· — reduction. At the same time we are able to rationalize a

structure-property-relationship upon the systematic investigation of a series of stable,

easily accessible and non-toxic mono- and tris-adducts II-VII of C60.[2c,4] This led to

the identification of new lcompounds for neuroprotective applications with

significantly improved superoxide dismutation activity.


101


Materials

A Chemicals: C60 was obtained from Hoechst AG/Aventis and separated from

higher fullerenes by a plug filtration.[5, 6] All chemicals were purchased by chemical

suppliers and used without further purification. All analytical reagent-grade solvents

were purified by distillation. Dry solvents were prepared using customary literature

procedures.[7] Thin layer chromatography (TLC): Riedel-de-Haën silica gel F254 and

Merck silica gel 60 F254. Detection: UV lamp and iodine chamber. Flash column

chromatography (FC): Merck 30 silica gel 60 (230–400 mesh, 0.04–0.063 nm). The

OO

OO

O OOO

O

O

O

O

OOO

OH

OHO

O

OH

O

ONO

OO

O OOO

O

O

O

O

OOO

N N

OO

OO

O OOO

O

O

O

O

O OHN

O OHN

OHN

OO

OHO

OHO

OHONH

OHO

OHO

HO

O

HOO

HO O OH

O

O OHN

O OOH

OOH

OO OH

O

O

O OHN

O OO

OO

OO O

O N

N

N

OHHO

OH

II

IV

III

V

VII

VI

HO

OHO

O

OHHOO O

OH

OHOO

I

3 Br

3 Br

Figure 5-1 structures of II-VII


102

HPLC-grade solvents were purchased from SDS or Acros Organics; analytical column

Nucleosil 5 μm, 200×4 mm, Macherey–Nagel, Düren. HPLC grade DMSO containing

a controlled amount of water (0.06 % after mixing in stopped-flow cuvette) was used

for the complex solutions, and the water content was determined by Karl Fischer

titration. Synthesis General Methods

Compound II, III, V, VI, VII and the precursor compound S1 were synthesized

according to literature procedure.[8] The synthesis flow chart of IV is shown in

Scheme 5-1.

OO

R1OOO H

N

OO

OO

O O

OO

R1OOO H

N

OHO

OHO

O OH

OO

R1OOO H

N

OO

OO

O O

Br

Br

BrOO

R1OOO H

N

OO

OO

O O

Br

Br

Br

OO

R1OOO H

N

OO

OO

O O

N

N

N

a)

b)

c)

d)

S1 S2

S3

S4

3 Br

III

a) formic acid, rt, 48 h; b) 2-bromoethanol, DCC, DMAP, 1-HOBt, THF, 0 °C → rt, 24 h; c) C60, CBr4, DBU, toluene, rt, 6 h; d) pyridine, 60 °C, two days

Scheme 5-1 the synthesis flow chart of IV


103

Synthesis of Malonate S2:

S1 (1.37 g, 1.58 mmol) was dissolved in formic acid (15 mL). The reaction mixture

was stirred for 48 h at room temperature and the progress of the reaction was

monitored by TLC. The reaction mixture was concentrated and dried in vacuum to

afford S2 as a white solid. (1.09 g, 1.58 mmol, 99 %). 1H-NMR (300 MHz, RT,

THF-d8): δ = 10.33 (s, br, 3H, COOH), 6.21 (s, br, 1H, CONH), 4.17 (t, 3J = 6.6 Hz,

2H, OCH2), 4.14 (t, 3J = 6.8 Hz, 2H, OCH2), 3.32 (s, 2H, OCCH2CO), 2.22 (t, 3J = 7.7

Hz, 6H, CH2COOH), 2.11 (t, 3J = 7.4 Hz, 2H, OCCH2), 1.98 (t, 3J = 7.8 Hz, 6H,

NHC(CH2)3), 1.67 (m, 6H, CH2), 1.25 (m, 32H, CH2), 0.91 (t, 3J = 6.7 Hz, 3H, CH3)

ppm. 13C-NMR (75 MHz, RT, THF-d8): δ = 174.88 (3C, COOH), 172.12 (1C, CONH),

166.87 (1C, CO), 166.79 (1C, CO), 65.66 (1C, OCH2), 65.31 (1C, OCH2), 57.19 (1C,

NHC(CH2)3), 41.44 (1C, OCCH2CO), 37.19 (1C, CH2CO), 31.86 (1C, CH2), 29.89 (3C,

NHC(CH2)3), 29.68, 29.64, 29.61, 29.49, 29.48, 29.37, 29.19 (10C, CH2), 28.71 (3C,

CH2COOH), 28.39, 28.18, 25.71 (5C, CH2), 25.48 (1C, CH2CH2CO), 25.18, 22.66 (2C,

CH2), 14.12 (1C, CH3) ppm. IR(ATR): ν;~ = 3402, 2963, 1871, 1648, 1507, 1482,

1467, 1457, 1432, 1324, 1266, 1117, 950, 791, 686, 624 cm-1. MS (FAB, NBA): m/z =

699 [M]+. C37H65NO11 · C2HF3O2: calcd. C 57.55, H 8.17, F 7.00, N 1.72, O 25.55;

found: C 57.99, H 8.52, N 2.01.


A solution of S2 (1.09 g, 1.56 mmol) and 2-bromoethanol (0.79 g, 6.32 mmol) in dry

THF (150 mL) was cooled to 0 °C under nitrogen atmosphere. DMAP (193 mg, 1.58

mmol), 1-HOBt (747 mg, 5.53 mmol) and DCC (1.14 g, 5.53 mmol) were added

subsequently. After stirring the solution under N2 for 2 h at 0 °C, it was left at room

temperature for another 24 h. Progress of the reaction was monitored by TLC. The

solution was filtered and after evaporation of the solvent the residue was dissolved in

ethyl acetate and filtered again for several times to remove the remaining DCU.

Purification was obtained by flash column chromatography (SiO2,

dichloromethane/ethyl acetate, 15:1 to 5:1). The purified material was dried in vacuum

affording S3 as a light yellow oil. (1.22 g, 1.20 mmol, 76 %). 1H-NMR (400 MHz, RT,


104

CDCl3): δ = 5.68 (s, br, 1H, CONH), 4.39 (t, 3J = 6.2 Hz, 6H, CH2CH2Br), 4.15 (t, 3J =

6.7 Hz, 2H, OCH2), 4.13 (t, 3J = 6.7 Hz, 2H, OCH2), 3.52 (t, 3J = 6.1 Hz, 6H, CH2Br),

3.37 (s, 2H, OCCH2CO), 2.36 (t, 3J = 7.8 Hz, 6H, CH2COO), 2.14 (t, 3J = 7.6 Hz, 2H,

OCCH2), 2.06 (t, 3J = 7.2 Hz, 6H, NHC(CH2)3), 1.64 (m, 6H, CH2), 1.25 (m, 32H,

CH2), 0.88 (t, 3J = 6.9 Hz, 3H, CH3) ppm. 13C-NMR (100 MHz, RT, CDCl3): δ =

172.73 (3C, COO), 172.35 (1C, CONH), 166.69 (1C, CO), 166.62 (1C, CO), 65.69

(1C, OCH2), 65.21 (1C, OCH2), 64.01 (3C, CH2CH2Br), 57.24 (1C, NHC(CH2)3),

41.54 (1C, OCCH2CO), 37.11 (1C, CH2CO), 31.86 (1C, CH2), 29.81 (7C, NHC(CH2)3,

CH2), 29.64, 29.47, 29.34 (6C, CH2), 28.77 (3C, CH2COO) , 28.58 (3C, CH2Br), 28.53,

28.29 (4C, CH2) , 25.91 (1C, CH2CH2CO), 25.63, 25.29, 22.80 (3C, CH2), 14.22 (1C,

CH3) ppm. IR(ATR): ν;~ = 3379, 2972, 2922, 1832, 1501, 1455, 1432, 1281, 1268,

1102, 950, 799, 791, 697, 686, 624 cm-1. MS (FAB, NBA): m/z = 1020 [M]+.

C43H74Br3NO11: calcd. C 50.60, H 7.31, Br 23.48, N 1.37, O 17.24; found: C 59.87, H

7.48, N 1.52.


C60 (741 mg, 1.03 mmol) was dissolved in dry toluene (ca. 0.5 mL toluene per mg C60)

under a nitrogen atmosphere. S3 (750 mg, 0.73 mmol) and CBr4 (342 mg, 1.03 mmol)

were added subsequently. DBU (153 μL, 1.03 mmol) in 20 mL toluene was added

dropwise over a period of 1 h to the stirred solution at room temperature. The reaction

mixture was stirred at room temperature for additional 6 h and the progress of the

reaction was monitored by TLC. The product was isolated by flash chromatography

(SiO2, toluene/ethyl acetate, 80:5 to 80:25) and dried in vacuum affording S4 as a red

brownish solid. (406 mg, 0.28 mmol, 32 %). 1H-NMR (400 MHz, RT, CDCl3): δ =

5.59 (s, br, 1H, CONH), 4.47 (t, 3J = 6.6 Hz, 4H, OCH2), 4.36 (t, 3J = 6.1 Hz, 2H,

CH2CH2Br ), 3.49 (t, 3J = 6.1 Hz, 6H, CH2Br), 2.33 (t, 3J = 7.6 Hz, 6H, CH2COO),

2.12 (t, 3J = 7.5 Hz, 2H, OCCH2), 2.03 (t, 3J = 7.3 Hz, 6H, NHC(CH2)3), 1.83 (m, 4H,

CH2), 1.67 (m, 2H, CH2), 1.45 (m, 2H, CH2), 1.25 (m, 30H, CH2), 0.85 (t, 3J = 7.0 Hz,

3H, CH3) ppm.


105

13C-NMR (100 MHz, RT, CDCl3): δ = 173.08 (3C, COO), 172.48 (1C, CONH),

164.00 (1C, CO), 163.92 (1C, CO), 145.65, 145.64, 145.55, 145.48, 145.47, 145.42,

145.17, 144.99, 144.96, 144.93, 144.89, 144.16, 143.40, 143.38, 143.31, 143.28,

143.26, 142.49, 142.15, 141.25, 141.22, 139.24, 138.14 (58C, C60-sp2), 71.84 (2C,

C60-sp3), 67.74 (1C, OCH2), 67.28 (1C, OCH2), 64.25 (3C, CH2CH2Br), 57.46 (1C,

NHC(CH2)3), 52.61 (1C, OCCCO), 37.32 (1C, CH2CO), 32.08 (1C, CH2), 29.87 (5C,

NHC(CH2)3, CH2), 29.82, 29.78, 29.77, 29.52, 29.38 (7C, CH2), 28.85 (3C, CH2Br),

28.75 (2C, CH2), 28.56 (3C, CH2COO), 28.47 (2C, CH2), 26.15 (1C, CH2CH2CO),

25.81, 25.27, 22.84, 21.59 (4C, CH2), 14.28 (1C, CH3) ppm. IR(ATR): ν;~ = 3332,

3062, 2992, 2634, 1832, 1766, 1703, 1654, 1603, 1533, 1478, 1403, 1281, 1235, 1107,

1033, 799, 787, 653 cm-1. MS (FAB, NBA): m/z = 1739 [M]+. UV/Vis (CH2Cl2): λmax

= 325.5, 425.5, 492 nm.

Synthesis of Malonate IV (PW85-cationic):

A solution of S4 (150 mg, 0.086 mmol) in 10 mL of dry pyridine was stirred for two

days at 60 °C. After the addition of 10 mL of toluene, the reaction mixture was

filtrated and the residue was suspended in toluene and distilled under vacuum for

several times to remove traces of pyridine. Reprecipitation from methanol/diethyl ether

gave IV as red brownish solid. (156 mg, 0.079 mmol, 92 %). 1H-NMR (400 MHz, RT,

DMSO-d6): δ = 9.13 (d, 3J = 5.6 Hz, 6H, o-PyrH), 8.65 (t, 3J = 7.8 Hz, 3H, p-PyrH),

8.20 (dd, 3J = 6.0, 7.7 Hz, 6H, m-PyrH), 7.20 (s, br, 1H, CONH), 4.91 (m, 6H,

CH2-Pyr), 4.51 (m, 10H, CH2CH2-Pyr, OCH2), 2.13 (m, 6H, CH2COO), 2.05 (m, 2H,

OCCH2), 1.73 (m, 6H, NHC(CH2)3), 1.48 (m, 2H, CH2), 1.35 (m, 4H, CH2), 1.17 (m,

32H, CH2), 0.84 (t, 3J = 6.2 Hz, 3H, CH3) ppm. 13C-NMR (100 MHz, RT, CDCl3): δ =

172.59 (3C, COO), 172.56 (1C, CONH), 162.92 (1C, CO), 162.90 (1C, CO), 146.33

(3C, p-PyrC), 145.56 (6C, o-PyrC), 145.51, 145.16, 144.95, 144.94, 144.89, 144.86,

144.80, 144.73, 144.52, 144.36, 144.34, 144.30, 144.25, 143.54, 143.51, 142.81,

142.74, 142.73, 141.85, 141.84, 141.53, 141.46, 140.67, 138.74, 138.22 (58C, C60-sp2),

128.21 (6C, m-PyrC), 71.55 (2C, C60-sp3), 67.37 (2C, OCH2), 62.56 (3C, CH2CH2-Pyr),

59.76 (3C, CH2-Pyr), 56.29 (1C, NHC(CH2)3), 52.70 (1C, OCCCO), 35.84 (1C,


106

CH2CO), 31.42 (6C, NHC(CH2)3), 29.18, 29.11, 29.01 (4C, CH2), 28.84 (3C,

CH2COO), 28.65, 28.57, 28.13, 27.80, 27.67 (7C, CH2), 25.69 (1C, CH2CH2CO),

25.08, 24.97, 22.22 (4C, CH2), 14.06 (1C, CH3). IR(ATR): ν;~ = 3368, 3020, 3001,

2651, 1799, 1765, 1654, 1613, 1546, 1434, 1406, 1256, 1249, 1237, 1111, 1023, 807,

753, 653, 603 cm-1. MS (FAB, NBA): m/z = 1897 [M]+, 908 [M]2+. UV/Vis

(DMSO/H2O): λmax = 258, 326 nm.

Instrumentation and Measurements see Chapter 2

Inderect arrays:

SOD activities of fullerenes were measured using the standard

McCord-Fridovich assay[9] based on ferricytochrome c reduction with superoxide

produced by xanthine/xanthine oxidase. The assay was performed at 25 °C in 3 mL of

reaction buffer (50 mmolL–1 potassium phosphate buffer, pH = 7.8) containing

ferricytochrome c (10 μmolL–1), xanthine (100 μM), and an amount of xanthine

oxidase such as to give a rate of ΔOD550nm ≈ 0.025 min–1 (about 0.01 U/mL) in the

absence of a putative SOD mimic. A reduction of ferricytochrome c was monitored at

550 nm. After 150 s, different amounts of the putative SOD mimic were added. Rates

were linear for at least 8 min. Both rates in the absence and in the presence of the

complex were determined for each concentration of complex added and plotted vs it.

The IC50 value represents the concentration of putative-SOD mimic that induces a 50%

inhibition of the reduction of cytochrome c.

To check that the tested compounds do not inhibit the production of superoxide

by xanthine oxidase, the rate of conversion of xanthine to urate (see below) was

determined by measuring the increase in absorbance at 290 nm over a 2-min period

with and without the tested compounds. To measure the rate of conversion of xanthine

to urate, xanthine oxidase (20 μL of 1 U/mL XO) was added to a solution of 50 mM

potassium phosphate buffer pH 7.8 containing xanthine (150 μmolL–1) at a final

volume of 1.0 mL at 25 °C. Urate production was monitored at 290 nm. No difference

in the slope was recorded with or without the putative SOD mimics.[10]


107

To further prove SOD activity of our fullerenes, a modified NBT assay was

used.[11, 12] In this assay an extensive excess of superoxide against catalyst is used. 1

mg of solid KO2 was added into 2 mL of 50 mM potassium phosphate buffer at pH 7.8

containing putative SOD mimetic and after 2 min spectra are recorded. NBT reacts

with superoxide forming the blue pigment formazan (lmax ≈ 580 nm (35 000 M-1 cm-1)).

The presence of complex caused concentration depending inhibition of formation, as

followed by the absorbance change at 580 nm. The concentration that causes 50% of

formation was indicated as IC50.

To detect the formation of hydrogen peroxide, product of superoxide

dismutation, solid KO2 was added into 25 mM solution of fullerens in 50mM

potassium phosphate buffer to a final concentration of 250 μM. Solutions were

incubated for 5 min at 370C and concentration of formed H2O2 determined. H2O2 was

quantified by measuring spectrophotometrically the coloured product formed by

peroxidase-catalyzed oxidation of 4-aminoantipyrine.[13] As a control, H2O2

formation after KO2 addition was measured in buffer without fullerenes. For

comparison the quantity of H2O2 was also followed in sample containing native

MnSOD (E. coli) (500 U/ml).



108

In order to describe the structure-reactivity-relationship with respect to

superoxide dismutase (SOD) activity, we first present the redox properties of II-VII.

Cyclic voltammetry measurements[14] in DMSO have shown that in the potential

0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0

6.0x10-6

3.0x10-6

0.0

-3.0x10-6

-6.0x10-6

-9.0x10-6

-1.2x10-5

-1.5x10-5

i [A

]

E [V] vs Ag/AgCl

0.0 -0.2 -0.4 -0.6 -0.8 -1.09.0x10-6

6.0x10-6

3.0x10-6

0.0-3.0x10-6

-6.0x10-6

-9.0x10-6

-1.2x10-5

-1.5x10-5

i [A

]

E [V] vs Ag/AgCl

II III

0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0

1.0x10-5

0.0

-1.0x10-5

-2.0x10-5

-3.0x10-5

-4.0x10-5

i [A

]

E [V] vs Ag/AgCl0.0 -0.2 -0.4 -0.6 -0.8 -1.0

5.0x10-6

0.0

-5.0x10-6

-1.0x10-5

-1.5x10-5

-2.0x10-5

-2.5x10-5

i [A

]

E [V] vs Ag/AgCl

IV V

0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -3.09.0x10-5

6.0x10-5

3.0x10-5

0.0

-3.0x10-5

-6.0x10-5

-9.0x10-5

-1.2x10-4

i [A

]

E [V] vs Ag/AgCl

0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.02.0x10-5

1.0x10-5

0.0

-1.0x10-5

-2.0x10-5

-3.0x10-5

-4.0x10-5

i [A

]

E [V] vs Ag/AgCl

VI VII

Figure 5-2 Cyclic voltammogramms of II-VII in DMSO purged with nitrogen. Conditions: [Fullerene] = 5 x 10-4 M, [Bu4NBF4] = 0.1 M, T = 298 K, scan rates = 0.2 V/s.


109

range from 0 to -1 V (vs. SCE) II-VII undergo two reversible reductions although VI

is not very clear. (Figure 5-2) The corresponding reduction potentials of the

monoadducts II-IV are significantly higher than those of tris-adducts V-VII and show

a prominent charge dependence especially for the first C60/C60·— redox couple with

the positively charged derivative IV being the strongest electron acceptor. The

observed redox potentials are considerably higher than what would be expected for

fullerene mono- and tris-adducts.[15] (Table 5-1) It seems that the amphiphilic nature

of the attached addends, facilitating micellar organization and therefore close C60-C60

interaction of II-VII in solution, is responsible for the positive shift of their redox

potentials. Of special importance is the fact that the first reduction potentials of II-IV

are much higher than the oxidation potential of superoxide (-0.74 V vs. SCE in

DMSO). This implies that the electron transfer from O2·— to those fullerenes

(Reaction I in Scheme 5-2) is not only strongly driven thermodynamically in DMSO,

but that it is also possible in aqueous solutions (E° (O2/O2·—) = -0.4 V vs. SCE in

water). Even in the case of the more difficult to reduce tris-adducts VI and VII this

process could be energetically feasible. It should be mentioned that photo-induced

C60·— is able to reduce O2 in aqueous solution, generating O2

·— in an outer-sphere

electron transfer process, due to the fact that its oxidation potential is more negative

(-0.56 V vs. SCE) than the reduction potential of O2,[16] and also more negative than

the corresponding potentials of II-VII. (Table 5-1)

Full + O2- Full + O2

-

-Full + O2- [ Full ]· O2

+H+

Full + H2O2

(I)

(II)- -

Full + O2- Full + O2

-

-Full + O2- [ Full ]· O2

+H+

Full + H2O2

(I)

(II)-

Full + O2- Full + O2Full + O2

-

-Full + O2- [ Full ]· O2[ Full ]· O2

+H+

Full + H2O2

(I)

(II)- -

Scheme 5-2


110

The cyclic voltammograms of II-V in dioxygen-saturated DMSO have revealed that in

the presence of these fullerenes the re-oxidation wave of superoxide (electrochemically

generated in situ) disappears. The current due to re-oxidation of the fullerene anions

diminishes, whereas the values of corresponding fullerene anions re-oxidation

potentials remain unaffected. (Figure 5-3) This behaviour clearly indicates that a

reaction between electrochemically generated superoxide and the fullerene anions

(Reaction II in Scheme 5-2) takes place without inducing chemical changes on the

fullerenes. The superoxide decomposition is also observed by applying much lower

(catalytic) concentrations of II-V, whereas VI and VII, independent on the applied

concentrations, do not affect the superoxide re-oxidation. These experiments suggest

not only that both II-V and their anions can react with O2·— but also that the reaction

has a catalytic character.

Fullerene 1E1/2a

(V)

2E1/2a

(V) IC50

b (μM) kMcCF x 106

(M-1s-1)

kcat x 106

(M-1s-1) Modified

NBT assayc

II -0.248 -0.614 2.11±0.02 1.3±0.2 2.64±0.04 +

III -0.224 -0.647 1.86±0.01 1.9±0.3 4.29±0.06 +

IV -0.077 -0.521 0.31±0.02 8.7±0.3 12.02±0.22 +

V -0.433 -0.726 2.85±0.03 0.9±0.2 0.26±0.02 +

VI -0.436 -0.732 d

VII -0.585 -1.094 d

a vs. SCE calibrated by the Fc+/Fc couple (0.43 V vs. SCE). bIC50 is the concentration of putative-SOD mimic that induces a 50% inhibition of the reduction of cytochrome c cThe NBT assay was qualitatively applied. dA precipitate was formed.

Table 5-1 Redox potentials, catalytic rate constants and IC50 values obtained by using direct stopped-flow measurements (kcat) in DMSO (0.06% water) and an indirect cytochrome c assay (kMcCF) in an aqueous solution (pH = 7.8), respectively


111

In order to address this point we further studied the reactions of II-VII with a large

excess of KO2 in DMSO containing a controlled amount of water (0.06%), which was

in excess over the superoxide and fullerene concentrations.[14] Time-resolved UV/Vis

spectra (Figure 5-4) have shown that immediately after mixing of a superoxide

solution with a fullerene solution rapid decomposition of O2·— (decrease of the

absorbance in the 240-330 nm range within the dead time of the stopped-flow

instrument) was observed in the case of II-V.[17] The products of superoxide

disproportionation, O2 and H2O2, were qualitatively detected in all four

experiments.[14] At much longer time scale the pyridinium groups of two cationic

dendrimers and VI react stoichiometrically with KO2 resulting in a product with the

strong absorbance at 273 nm. The corresponding unattached malonate addends

themselves, as well as VI and VII do not induce superoxide decomposition.[18] The

rapid process was quantified by following the corresponding absorbance decrease at

270 nm in a series of stopped-flow measurements, in which the catalytic concentration

of the studied fullerenes was varied. Application of a microcuvette accessory, which

reduced the dead time of the instrument down to 0.4 ms, enabled the observation of the

fast disappearance of the 270 nm absorption. This behavior could best be fitted as a

-1.5 -1.0 -0.5 0.0

-6.0x10-5

-4.0x10-5

-2.0x10-5

0.0

2.0x10-5

4.0x10-5

i / A

E/V vs SCE

DMSO purged with O2 II in DMSO purged with N2 II in DMSO purged with O2

Figure 5-3 Cyclic voltammogramms of II purged with nitrogen and oxygen, and of pure DMSO purged with oxygen. Conditions: [II] = 0.5 x 10-3 M, [Bu4NBF4] = 0.1 M, T = 298 K, scan rates = 0.2 V/s.


112

first-order process to obtain the characteristic kobs (s-1) value. A good linear correlation

between kobs and the fullerene concentration was observed and from the slopes of the

corresponding plots the catalytic rate constants (kcat.)[14, 19] were determined. (Figure

5-5, Table 5-1)

This is the first time that the catalytic SOD activity of fullerenes has been detected by

using a direct method. Thereby, also interference with additional components that are

always present in indirect assays can be ruled out. Importantly, our results show a clear

dependence of the fullerenes SOD activity (kcat) on their reduction potentials, namely,

the higher the reduction potential (ability to be reduced by O2·—), the higher the SOD

activity. (Table 5-1) This suggests that the electron transfer from O2·— to the fullerene

plays the key role in the overall catalytic dismutation of superoxide. At the same time,

although VI has almost the same reduction potential as V, it does not induce catalytic

decomposition of O2·—, which additionally implies that the architecture of fullerene

derivatives also plays an important role. In contrast to V, the capped structure of VI

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

250 300 350 400 450 5000.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

Abso

rban

ce

Wavelength(nm)

Abs

orba

nce

Wavelength (nm)

A

B

Figure 5-4 Time resolved UV/vis spectra recorded for the reaction of II (5 x 10-5 M) with 1 mM KO2 in DMSO at room temperature. A: spectrum recorded (measurements in tandem cuvette) before mixing; B: first spectrum obtained after mixing (using a stopped-flow module) followed by spectra recorded at time intervals of 10 s (total observation time 30 min). Inset: control reaction without addition of the fullerene followed over 2.5 h.


113

most probably does not allow for a favorable attractive interaction between O2·— and

the functionalized region of C60 cage, which could include H-binding with the three

OH-groups of the malonate addends. Such an interaction, however, could be essential

for the superoxide reduction and stabilization of the resulting peroxide in the second

step of the SOD cycle (as it was observed in the case of metal based SOD

mimetics[20]).

Although widely used indirect SOD assays are not very reliable in the case of enzyme

mimetics (the direct stopped-flow method is a better probe for a SOD activity even

though it requires a non-aqueous medium),[14, 19] for comparison we also applied

cytochrome c[21] (Figure 5-6a) and modified NBT assays[14] to investigate the SOD

activity of our fullerenes in an aqueous buffer in a manner utilized in the literature. The

trisadducts VI and VII show no SOD activity, whereas II-V [22] exhibit the SOD

activity with the catalytic rate constants (kMcCF)[14] reflecting the same trend as that

determined by the direct stopped-flow measurements. (Table 5-1) The time resolved

UV/vis spectra for the reaction between electrochemically reduced cytochrome c and

0.0 2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-5 1.0x10-40

100

200

300

400

500

600

k obs [

s-1]

cfullerene [M]

II III IV V

Figure 5-5 Plots of kobs

versus [fullerene] for the reaction between fullerenes and saturated KO2 in DMSO

solution at room temperature


114

the fullerenes under an argon atmosphere were monitored over 30 min and 12 h. No

change in the redox state of cyt cII was observed. None of the fullerenes interfered with

the cytochrome c assay by reoxidizing cyt cII to cyt cIII. However, in the entire spectral

range a slow shift of the cytochrome c spectrum have been observed in the case of VI

and VII, suggesting that these fullerenes induce a precipitation of cytochrome c from

the solution. This attributes to the somewhat higher value of apparent kcat for VII in

comparison to the value obtained in the stopped-flow experiment, by causing an

additional decrease of the absorbance at 550 nm. (Figure 5-7)

A lower solubility of II-IV in water caused the somewhat smaller kMcCF compared with

kcat determined in DMSO. Upon using an excess of superoxide over the putative SOD

mimetic we also determined the formation of H2O2.[24] The SOD active fullerenes

II-V yield more H2O2 as compared with the amount produced by spontaneous

dismutation of superoxide in the aqueous buffer, in a similar fashion as the native

enzyme. (Figure 5-6b) The amount of produced H2O2 correlated with the fullerene

SOD activity. The capped trisadduct VI and VII had no effect on H2O2 formation,

proving again their lack of SOD activity.

350 400 450 500 550 600 650 7000.0

0.5

1.0

1.5

2.0

2.5

0 200 400 600 800 1000120014001600 18000.38

0.40

0.42

0.44

0.46

0.48

0.50

0.52

0.54

0.56

0.58

Abs

orba

nce

time, s

550 nm,

abso

rban

ce [a

.u.]

wavelength [nm]

Cyt CII with 7

Figure 5-7 The time resolved UV/Vis spectra for the reaction between electrochemically reduced cytochrome c and VII under an argon atmosphere (50 mM potassium phosphate buffer, pH = 7.8; 25 °C; [cyt c] = 10 μM and [VII] =20 μM). Inset: corresponding absorbance change with time at 550 nm.


115

5.5 Conclusion

In conclusion we have shown by electrochemical, spectrophotometrical and

sub-millisecond mixing UV/Vis stopped-flow measurements, as well as by indirect

SOD assays and H2O2 detection that II-V act as SOD mimetics. For the first time we

have demonstrated that there is a direct correlation between SOD activity of the water

soluble fullerenes and molecular properties such as i) reduction potential, ii) charge

and iii) molecular structure. Monoadducts II-IV of C60 have been identified as very

active new lead structures. Even more, the positively charged monoadduct IV, is by

one order of magnitude more active than C3 (I) and approaches the performance of

natural Mn- and Fe-SODs.[3a] Its activity is comparable with that of the highly active

metal containing SOD mimics.[14] In addition to the high activity the monoadducts

have a number of advantages over C3 (I), since they are: i) more stable, ii)

considerably less toxic[2d] and iii) can easily be produced in large quantities. Knowing

that upon irradiation solubilized C60, its aggregates and some water soluble derivatives

can generate superoxide[16] (on which their potential application in photodynamic

0 20 40 60 80 100 120 140 160 1800.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

A55

0 (A

U)

Time (s)

control 3.33 μM III 3.33 μM V 0.83 μM II 0.83 μM IV

Figure 5-6 a) Kinetics of the reduction of ferricytochrome c (550 nm) without and with the fullerenes

at room temperature; b) Production of H2O2 by SOD (25 μM) and fullerenes (25 μM) from KO2 (250 μM;

pH = 7.8; 37 °C).


116

therapy is based), it is crucial to understand how fullerene derivatives should be

designed in order to either decompose or generate O2·—, depending on a direction of

their biomedical application. In that light our results will certainly be of fundamental

importance.

5.6 Note and References

[1] I. Lamparth, A. Hirsch, J. Chem. Soc., Chem. Commun.，1994, 1727-1728.

[2] a) L. L. Dugan, E. G. Lovett, K. L. Quick, J. Lotharius, T. T. Lin, K. L.

O’Malley Parkinsonism Relat. Disord. 2001, 7, 243-246; b) L. L. Dugan, D.

M. Turetsky, C. Du, D. Lobner, M. Wheeler, C. R. Almli, C. K. Shen, T. Y.

Luh, Proc. Natl. Acad. Sci. USA 1997, 94, 9434-9439; c) P. Witte, F. Beuerle,

U. Hartnagel, R. Lebovitz, A. Savouchkina, S. Sali, D. Guldi, N. Chronakis,

A. Hirsch, Org. Biomol. Chem., 2007, 5(22), 3599-3613; d) F. Beuerle, P.

Witte, U. Hartnagel, R. Lebovitz, C. Parng, A. Hirsch, Journal of

Experimental Nanoscience, 2007, 2(3), 147-170.

[3] a) S. S. Ali , J. I. Hardt, K. L. Quick, J. S. Kim-Han , Erlanger, F. Bernard,

T.-T. Huang, C. J. Epstein and L. L. Dugan, Free Rad. Biol. Med., 2004, 37,

1191-1202; b) K. Okuda, T. Mashino, M. Hirobe, Bioorg. Med. Chem. Lett.

1996, 6, 539-542.

[4] F. Beuerle, N. Chronakis and A. Hirsch, Chem. Commun., 2005, 3676-3678.

[5] U. Reuther, Ph. D. Dissertation, University of Erlangen-Nuremberg,

Germany, 2002.

[6] L. Isaacs, A. Wehrsig, F. Diederich, Helv. Chim. Acta 1993, 76, 1231–1250.

[7] D. D. Perrin. W. L. F. Amarego, Purification of Laboratory Chemicals, 3rd

ed., Pergamon Press, Oxford, 1988.

[8] P. Witte, F. Beuerle, U. Hartnagel, R. Lebovitz, A. Savouchkina, S. Sali, D.

Guldi, N. Chronakis, A. Hirsch, Org. Biomol. Chem., 2007, 5(22),

3599-3613.

[9] J. M. McCord and I. Fridovich, J. Biol. Chem. 1969, 244, 6049-6055.


117

[10] E. F. Elstner and A. Heupel, Anal. Biochem. 1976, 70, 616-620.

[11] M. Sun and S. Zigman, Anal. Biochem. 1978, 90, 81-89.

[12] R. E. Heikkila and F. Cahhat, Anal. Biochem. 1976, 75, 356-362.

[13] Ioannidis, I. and de Groot, H. Biochem. J., 1993, 296 (Pt 2), 341-345.

[14] G.-F. Liu, M. Filipović, F. W. Heinemann, I. Ivanović-Burmazović, Inorg.

Chem. 2007, 46, 8825-8835. For the experimental details regarding solution

preparations, instrumentation techniques.

[15] C. Boudon, J.-P. Gisselbrecht, M. Gross, L. Isaacs, H. L. Anderson, R. Faust,

F. Diederich, Helv. Chim. Acta 1995, 78(5), 1334-44.

[16] a) I. Nakanishi, S. Fukuzumi, T. Konishi, K. Ohkubo, M. Fujitsuka, O. Ito, N.

Miyata, J. Phys. Chem. B 2002, 106, 2372-2380; b) Y. Yamakoshi, N.

Umezawa, A. Ryu, K. Arakane, N. Miyata, Y. Goda, T. Masumizu, T.

Nagano, J. Am. Chem. Soc. 2003, 125, 12803-12809.

[17] Less prominent spectral changes at the wavelengths higher than 350 nm, in

the case of II and III, were found to be caused by KOH which is inevitably

present in KO2. By using an electrochemically generated superoxide solution

these spectral changes were not observed.

[18] The same product is formed upon reaction with KOH.

[19] a) D. P. Riley, W. J. Rivers, R. H.Weiss, Anal. Biochem. 1991, 196, 344-349;

b) R. H. Weiss, A. G. Flickinger, W. J. Rivers, M. M. Hardy, K. W. Aston, U.

S. Ryanll, D. P. Riley, J. Biol. Chem. 1993, 268 (31), 23049.

[20] D. T. Sawyer, J. S. Valentine, Acc. Chem. Res. 1982, 14, 393-400.

[21] J. M. McCord, I. Fridovich, J. Biol. Chem. 1969, 244, 6049-6055.

[22] V has quite low, but still detectable SOD activity.


118

Summary

This thesis includes work on:

[1] Syntheses and structural characterization of related seven-coordinate complexes

with different set of donor atoms.

[2] Kinetic and thermodynamic investigations of the reactions of superoxide radical

with our new class of potential seven-coordinate SOD active iron and manganese

complexes.

[3] A new sub-millisecond mixing UV/Vis stopped-flow measurement method for the

investigation of SOD activity was developed and compared with traditional indirect

SOD assays.

[4] A reactivity towards superoxide of series of stable, easily accessible and non-toxic

derivatives of C60 was investigated by electrochemical, spectrophotometrical and

submillisecond mixing UV/Vis stopped-flow measurements, as well as by indirect

SOD assays and H2O2 detection.

Although indirect assays based on the cytochrome c assay have been developed

and used to measure SOD activity in the literature, our results have shown that they are

not very reliable. They can be applied only upon considering possible cross reactions

between indicator substance and the studied complex in their different oxidation forms,

in which they may occur within the SOD catalytic cycle. The direct stopped-flow

method, where the high excess of superoxide over complex can be utilized, is a better

probe for a complex SOD activity.

Seven-coordinate 3d metal complexes gained a vast recognition as the synthetic

superoxide dismutase enzymes (SODs) in biological systems. A number of recent

inventions, patented by the companies which are involved in the development of

Summary

119

metal-based therapeutics and in businesses of improving the quality of life, are devoted

to the application of iron and manganese seven-coordinate complexes as human

medicaments. Therefore, it is of interest to bring the chemistry of the seven-coordinate

3d metal complexes to a level of full understanding of their structural and solution

properties and to conceive whether and how metal complexes can be used as

pharmaceuticals for treatment of disease states caused by superoxide overproduction.

To this goal a series of seven-coordinate manganese and iron complexes were

synthesized and their SOD activity was evaluated by the electrochemical,

spectrophotometrical and sub-millisecond mixing UV/Vis stopped-flow measurements,

as well as by indirect SOD assays.

Although it has been postulated in the literature that only seven-coordinate

complexes of macrocyclic ligands with prominent conformational flexibility could

possess SOD-activity, our seven-coordinate iron and manganese complexes

[FeIII(dapsox)(H2O)2]ClO4·H2O (1), [FeII(H2dapsox)(H2O)2](NO3)2·H2O (2) and

[MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2(H2O) (3) with the acyclic and rigid

H2dapsox ligand demonstrate ability for catalytic decomposition of superoxide. The

demonstrated SOD activity of these rigid seven-coordinate complexes proves that

water release and formation of a six-coordinate intermediate, requiring conformational

rearrangement of the ligand, is not the rate-limiting step in the overall inner-sphere

catalytic SOD pathway of the proven macrocyclic SOD mimetics. Furthermore, it also

shows that conformational flexibility of the pentadentate ligand is not the key factor

assisting SOD activity, and that the acyclic and rigid ligand systems can also be

considered as structural motifs for designing SOD mimetics.

The important role of a ligand electronic properties and amido groups in

stabilizing the FeIII oxidation state in six-coordinate geometry led us to gain further

insight on the effect of the amide group on the redox behavior of manganese

complexes. Therefore, we studied the corresponding MnII complex

[MnII(Dcphp)(CH3OH)2](CH3OH)2 (5) with two hydrazido nitrogens in the


120

seven-coordinate geometry coordination and its reactivity towards O2•–. The results

show the strong σ-donor ability of negatively charged hydrazido nitrogen in the first

coordination sphere of the manganese center significantly decreases MnIII/MnII redox

potential by stabilizing MnIII form of the complex, and as a result, this complex

catalyzes the fast disproportionation of superoxide under the applied experimental

conditions. Whereas the complex [MnII(Daphp)(H2O)2](ClO4)2 (6) does not show

SOD activity because of the lack of the carbonyl groups in Daphp, which makes it less

acidic, resulting in its coordination in the neutral hydrazone form and corresponding

redox potential doesn’t fall between the redox potentials for the reduction and

oxidation of O2·– under applied experimental conditions.

In order to demonstrate existence of eight-coordinate MnII species, such a

complex [MnII(Hdapmp)2](ClO4)2(H2O)2 (7) was synthesized and characterized. At

the same time, by studying the reaction of this coordination saturated and consequently

substitution inert manganese species with superoxide, we could further probe whether

SOD catalysis can be achieved by an outer sphere mechanism, which has been

proposed as a parallel pathway operating within the seven-coordinate MnII SOD

mimetics. The results show that the studied eight-coordinated MnII complex

demonstrates no ability for catalytic decomposition of superoxide although the redox

potential of the corresponding MnIII/MnII couple is similar to the redox potentials of

the proven SOD mimetic [MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2(H2O) (3). This

point out that the redox potential is not the only important requirement for a complex

to be the efficient SOD mimetic. The efficient catalysis seems to be facilitated by

binding of superoxide to the redox-active metal center within an inner-sphere electron

transfer mechanism.

There is one phenomenon, which has been reported in the literature, that the Fe

and Mn catalysts lose their SOD activity when their concentrations increase in the SOD

assays. In order to explain this phenomenon, regarding structural features that might

have an influence on a complex reactivity toward superoxide, we synthesized a

Summary

121

seven-coordinate μ-oxo-dimer [(H2O)2FeIII2(Daphp)2O](ClO4)4 (8) and also tested the

reactivity of this complex toward superoxide. We could demonstrate that a

stoichiometric two step reaction is possible for this dimer complex, which most

probably involves substitution of the labile solvent molecules at two iron centers when

reacting with superoxide. These results confirmed the literature postulation that

oxo-dimmer structures do not have SOD catalytic activity, so the dimer formation

becomes favored when the concentration of complex increases, thereby lowering the

apparent catalytic activity.

Another class of compounds that has been considered for efficient elimination

of the superoxide radicals is the calls of water-soluble fullerenes. In order to describe

the structure-reactivity-relationship with respect to superoxide dismutase (SOD)

activity and rationalize this relationship upon the systematic investigation, a series of

stable, easily accessible and non-toxic mono- and derivatives of C60 (II-IIV) were

synthesized and their SOD activity was evaluated. By way of comparison, we present

for the first time clear and unambiguous evidence for a catalytic dismutation process

whose key steps are successive O2·— oxidation, within an outer sphere electron transfer

processes, and fullerene derivative mediated O2·— reduction. For the first time we have

demonstrated that there is a direct correlation between SOD activity of the water

soluble fullerenes and molecular properties such as i) reduction potential, ii) charge

and iii) molecular structure.


122

Zusammenfassung

In dieser Arbeit wurden folgende Themengebiete behandelt:

[1] Die Synthesen und strukturelle Charakterisierung von verwandten siebenfach

koordinierten Komplexen mit unterschiedlichen Sätzen an Donoratomen.

[2] Kinetische und thermodynamische Untersuchungen der Reaktionen des

Superoxidradikalanions mit unserer neuen Klasse an potentiellen siebenfach

koordinierten SOD aktiven Eisen- und Mangankomplexen, sowie Untersuchungen der

verwandten Substitutions- und Elektronentransferprozesse in verschiedenen

Lösungsmitteln.

[3] Eine neueUV/vis stopped-flow Meßmethode mit Mischzeiten unter einer

Millisekunde wurde entwickelt und mit herkömmlichen indirekten SOD Assays

verglichen.

[4] Eine Serie von stabilen, einfach zu bekommenden und ungiftigen C60-Derivaten

wurden synthetisiert und sowohl mittels elektrochemischer, spektrophotometrischer

und UV/vis stopped-flow Messungen als auch mit indirekten SOD Assays und

H2O2-Messungen untersucht

Obwohl viele indirekte Assays, basierend auf dem Cytochrom c Assay in der

Literatur, bekannt sind und verwendet werden, um die SOD-Aktivität zu messen,

haben unsere Forschungsergebnisse gezeigt, dass diese nicht verlässlich sind, sie

können nur verwendet werden, wenn man mögliche Reaktionen zwischen der

Indikatorsubstanz und dem untersuchten Komplex in ihren verschiedenen

Oxidationsstufen, in denen sie im SOD Katalysezyklus auftreten, in Betracht zieht. Die

direkte stopped-flow-Methode, wo ein hoher Überschuß an Superoxid gegenüber dem

Komplex verwendet werden kann, ist ein besserer Maßstab für die SOD-Aktivität

eines Komplexes.

Zusammenfassung

123

Nachdem sich eine Klasse von siebenfach koordinierten 3d Komplexen als

synthetische Superoxiddismutase-Enzyme (SODs) in biologischen Systemen nach und

nach durchgesetzt hat, wurden eine Reihe von Erfindungen von Firmen patentiert, die

bereits in die Entwicklung von metallbasierten Pharmazeutica eingebunden waren, um

die Chemie der siebenfach koordinierten 3d-Metallkompolexe und ihre strukturellen

Eigenschaften und ihr Verhalten in Lösung vollständig zu verstehen. So kann vielleicht

geklärt werden, ob und wie Metallkomplexe als Pharmazeutika für die Behandlung

von durch eine Überproduktion von Superoxid verursachten Krankkeiten eingesetzt

werden können. Eine Reihe von siebenfach koordinierten Mangan- und

Eisenkomplexen wurden synthetisiert und sowohl mittels elektrochemischer,

spektrophotometrischer und UV/vis stopped-flow Messungen als auch mit indirekten

SOD Assays auf ihre SOD-Aktivität hin untersucht.

Obwohl in der Literatur postuliert wurde, dass nur siebenfach koordinierte

Komplexe makrozyklischer Liganden mit einer ausgeprägten Flexibilität

SOD-Aktivität aufweisen können, zeigen unsere siebenfach koordinierten Eisen- und

Mangankomplexe [FeIII(dapsox)(H2O)2]ClO4·H2O (1),

[FeII(H2dapsox)(H2O)2](NO3)2·H2O (2) und

[MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2(H2O) (3) mit dem acylischen und starren

H2dapsox-Liganden die Fähigkeit zur katalytischen Zersetzung von Superoxid, was

auf ihre Fähigkeit zur Deprotonierung des koordinierten mehrzähnigen Liganden und

Änderung der pentagonal bipyramidalen Geometrie des Koordinationszentrums

zurückzuführen ist, was für die Bindungslängen und –winkel entscheidend ist. Die

nachgewiesene SOD-Aktivität dieser starren siebenfach koordinierten Komplexe

beweist, dass die Wasserabspaltung und die Bildung eines sechsfach koordinierten

Intermediates, die eine konformelle Umstrukturierung des Liganden erfordert, nicht

der geschwindigkeitsbestimmende Schritt im inner-sphere-Mechanismus des SOD

Katalysezyklusses sein kann. Darüberhinaus zeigt dies, dass die konformelle

Flexibilität des fünfzähnigen Liganden nicht der Schlüssel zur SOD-Aktivität ist, und


124

dass auch acyclische und starre Ligandsysteme für die Entwicklung von SOD

Mimetika in Frage kommen.

Der Amidgruppe kommt bei der Stabilisierung von FeIII in einer sechsfach

koordinierten Geometrie eine wichtige Rolle zu, was uns dazu gebracht hat, den Effekt

der Amidgruppe auf das Redoxverhalten von Mangankomplexen zu untersuchen. Wir

haben deshalb den korrespondierenden MnII-Komplex

([Mn(Dcphp)(CH3OH)2](CH3OH)2) mit zwei hydrazidischen Stickstoffatomen in der

siebenfach koordinierten Geometrie und seine Reaktivität gegenüber O2•– untersucht.

Die Ergebnisse zeigen, dass die starken σ-Donoreigenschaften der negativ geladenen

hydrazidischen Stickstoffatome in der ersten Koordinationssphäre des Mangans das

MnII / MnIII – Redoxpotential stark absenken, also die MnIII-Form des Komplexes

stabilisieren, wodurch der Komplex die schnelle Disproportionierung von Superoxid

unter den verwendeten experimentellen Bedingungen katalysiert; wohingegen der

Komplex MnII(Daphp)(H2O)2](ClO4)2 keine SOD-Aktivität zeigt, da in Daphp die

Carbonylgruppen fehlen, was den Liganden weniger azide macht und zu einer

Koordination in der neutralen Hydrazonform führt, so dass das Redoxpotential des

Komplexes nicht zwischen die Redospotentiale für die Reduktion und die Oxidation

von O2•– fällt.

Um die Existenz von achtfach koordinierten MnII-Spezies nachzuweisen, habe

ich den Komplex [MnII(Hdapmp)2](ClO4)2(H2O)2 synthetisiert und charakterisiert.

Durch die Untersuchung der Reaktion zwischen diesem abgesättigtem und daher

inerten Mangankomplex mit Superoxid, können wir einen näheren Einblick gewinnen,

inwiefern die SOD Katalyse durch einen outer-sphre-Mechanismus erreicht werden

kann, der als paralleler Reaktionspfad innerhalb des Katalysezyklusses der siebenfach

koordinierten MnII SOD-Mimetika vorgeschlagen wurde. Die Ergebnisse zeigen, dass

dieser achtfach koordinierte MnII Komplex keine Fähigkeiten für eine katalytische

Zersetzung von Superoxid zeigt, obwohl das Redoxpotential des MnIII / MnII-Paares

mit dem des bekannten SOD-Mimetikums

Zusammenfassung

125

[MnII(H2dapsox)(CH3OH)(H2O)](ClO4)2(H2O) (3) vergleichbar ist. Dies zeigt, dass

das Redoxpotential nicht die einzige nötige Bedingung für die SOD-Aktivität ist. Eine

effiziente Katalyse scheint durch die Bindung von Superoxid zum redoxaktiven

Metallzentrum innerhalb eines inner-sphere Elektronentransfermechanismusses

erleichtert zu werden.

Um das Phänomen, dass der Katalysator Aktivität verliert, wenn die

Konzentration von Eisen- oder Mangan-SOD-Katalysator erhöht wird, zu erklären,

haben wir den siebenfach koordinierten dimeren μ-oxo-Komplex

[(H2O)2FeIII2(DapHp)2O](ClO4)4 synthetisiert, um die Einflüsse möglicher

struktureller Eigenschaften dieses Komplexes und seine Reaktion mit Superoxid zu

untersuchen. Wir konnten zeigen, dass eine stöchiometrische zweistufige Reaktion

ablaufen kann, die wahrscheinlich die Substitution der labilen Solvensmoleküle durch

Superoxid an den zwei Eisenzentren beinhaltet. Diese Messergebnisse bestätigen das

Postulat in der Literatur, wonach Oxodimere keine SOD-Aktivität aufweisen, so dass

gefolgert werden kann, dass die Bildung der Dimeren gefördert wird, wenn die

Konzentration an Komplex erhöht wird und sich dadurch die katalytische Aktivität

erniedrigt.

Einige Veröffentlichungen haben berichtet, dass wasserlösliche Fullerene eine

exzellente Effizienz in der Zerstörung von Superoxidradikalen aufweisen. Um die

Struktur-Reaktivitäts-Beziehungen bezugnehmend auf die SOD-Aktivität zu

beschreiben und dieses Verhalten zu systematisieren, wurde eine Serie von stabilen,

einfach zugänglichen und ungiftigen C60-Derivaten synthetisiert und sowohl mittels

elektrochemischer, spektrophotometrischer und UV/vis stopped-flow Messungen als

auch mit indirekten SOD Assays und H2O2-Messungen untersucht. Wir zeigen zum

ersten Mal einen klaren und unzweideutigen Beweis für einen katalytischen Prozess,

dessen Schlüsselschritte eine sukzessive O2•–-Oxidation über einen outer-sphere

Elektronentransferprozess und die durch das Fullerenderivat vermittelte

O2•–-Reduktion sind. Zum ersten Mal haben wir gezeigt, dass es eine direkte


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Korrelation zwischen der SOD-Aktivität der wasserlöslichen Fulleren und molekularer

Eigenschaften, wie i) Redoxpotential, ii) Ladung und iii) molekularer Struktur gibt.

Curriculum Vitae

127

Curriculum Vitae

Personal details

Name: Gao-Feng Liu

Date of birth: 29. 05. 1975

Martial status: Married

Place of birth: Hunan, China

Nationality: Chinese

• Education and Qualifications 2003- 2008 Ph. D. studies in department of chemistry and pharmacy, university

of Erlangen-Nürnberg in Germany, supervisor: Prof. Dr. Dr. h. c. Rudi van Eldik and Dr. Ivana Ivanović-Burmazović, title “Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics”.

1999 - 2002 Msc. study in department of chemistry, Sun Yat-Sen university in China, supervisor: Prof. Dr. Xiao-Ming Chen, title “the design, synthesis, property and topology study of series of metal dicarboxylate coordination polymers”.

1994 - 1998 Bsc. study in department of chemistry, Nanjing University of

Science & Technology, title “The physical property improvement of macromolecule polymer by adding nanometer TiO2 particles technique”.

• Work Experience

02. 2003. - 07. 2002 Visit Researcher in Prof. Hai-Liang Zhu’s group, Chemistry

Department of Wuhan National University 06. 1999. - 07. 1998 Quality Inspector in a ShuangFeng International Business and

Economics Ltd.

• Publication


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1 Gao-Feng Liu, Ralph Puchta, Frank W. Heinemann, Ivana

Ivanović-Burmazović, Chemical Communication (Cambridge, United

Kingdom) (Submitted)

2 Gao-Feng Liu, Miloš Filipović, Ivana Ivanović-Burmazović, Florian Beuerle,

Patrick Witte, Andreas Hirsch, Angewandte Chemie (in print)

3 Gao-Feng Liu, Miloš Filipović, Frank W. Heinemann and Ivana

Ivanović-Burmazović, Inorganic Chemistry (2007), 46, 8825-8835.

4 David Sarauli, Roland Meier, Gao-Feng Liu, Ivana Ivanovic-Burmazovic, Rudi

Van Eldik, Inorganic Chemistry, (2005), 44(21), 7624-7633.

5 Yan-Zhen Zheng, Gao-Feng Liu, Bao-Hui Ye, Xiao-Ming Chen, Zeitschrift

fuer Anorganische und Allgemeine Chemie, (2004), 630(2), 296-300.

6 Ling-Yun Zhang, Gao-Feng Liu, Shao-Liang Zheng, Bao-Hui Ye, Xian-Ming

Zhang, Xiao-Ming Chen, European Journal of Inorganic Chemistry, (2003),

(16), 2965-2971.

7 Hai-Liang Zhu, Xian-Ming Zhang, Xiu-Ying Liu, Xian-Jiang Wang, Gao-Feng

Liu, Anwar Usman, Hoong-Kun Fun, Inorganic Chemistry Communications,

(2003), 6(8), 1113-1116.

8 Hai-Liang Zhu, Xian-Ming Zhang, Gao-Feng Liu, Da-Qi Wang, Zeitschrift

fuer Anorganische und Allgemeine Chemie, (2003), 629(6), 1059-1062.

9 Jin-Hua Yang, Shao-Liang Zheng, Jun Tao, Gao-Feng Liu, Xiao-Ming Chen,

Australian Journal of Chemistry, (2002), 55(11), 741-744.

10 Xiao-Ming Chen, Gao-Feng Liu, Chemistry--A European Journal (2002),

8(20), 4811-4817.

11 Gao-Feng Liu, Bao-Hui Ye, Yong-Hua Ling, Xiao-Ming Chen, Chemical

Communications (Cambridge, United Kingdom) (2002), (14), 1442-1443.

12 Gao-Feng Liu, Zheng-Ping Qiao, He-Zhou Wang, Xiao-Ming Chen, Guang

Yang, New Journal of Chemistry, (2002), 26(6), 791-795.

13 Guang Yang, Gao-Feng Liu, Shao-Liang Zheng, Xiao-Ming Chen, Journal of

Coordination Chemistry, (2001), 53(3), 269-279.

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