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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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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)
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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]
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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),
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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)
Chapter 1 Introduction
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 1 Introduction
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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)
Table 2-4 selected bond lengths (Å) and bond angles (deg) of 3
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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)
Table 2-6 selected bond lengths (Å) and bond angles (deg) of 4
Scheme 2-3 the possible mechanism of our seven coordinate Fe or Mn complexes for the decomposition of superoxide
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
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,
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[4] (a) D. Salvemini, D. P. Riley, S. Cuzzocrea, Nat. Rev. Drug Discovery 2002, 1,
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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.
Misko, M. G. Currie, S. Cuzzocrea, J. A. Sikorski, D. P. Riley, Science 1999,
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.
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[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.
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Grodkowski, P. Neta, I. Fridovich, Inorg. Chem. 2001, 40, 726-739. (g) S.
Yamaguchi, A. Kumagai, Y. Funahashi, K. Jitsukawa, H. Masuda, Inorg.
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2006, 45, 2358–2360. (j) S. Durot, F. Lambert, J.-P. Renault, C. Policar, Eur.
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[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.
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[9] D. P. Riley, W. J. Rivers, R. H. Weiss, Anal. Biochem. 1991, 196, 344–349.
[10] 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.
[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,
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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.
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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.
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[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.
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[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.
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[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.
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[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.
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[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
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[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.
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[31] Detailed kinetic investigations are in progress.
[32] D. P. Riley, Adv. Inorg. Chem. 2007, 59, 233–263.
Chapter 2 Seven-Coordinate Iron and Manganese Complexes with Acyclic and Rigid Pentadentate Chelates and their Superoxide Dismutase Activity
59
[33] D.-H. Chin, G., Jr. Chiericato, E. J., Jr. Nanni, D. T. Sawyer, J. Am. Chem.
Soc. 1982, 104, 1296-1299.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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]
3.3 Experimental Section
Materials and Instrumentation and Measurements see Chapter 2 Preparation of 2,6-bis(methoxycarbonyl)pyridine
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
66
3.4 Results and Discussion
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).
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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)..
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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).
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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]+
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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+
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Misko, M. G. Currie, S. Cuzzocrea, J. A. Sikorski, D. P. Riley, Science 1999,
286, 304.
[4] D. P. Riley, Chem. Rev. 1999, 99, 2573.
[5] C. Muscoli, S. Cuzzocrea, D. P. Riley, J. L. Zweier, C. Thiemermann, Z.-Q.
Wang, D. Salvemini, Br. J. Pharmacol. 2003, 140, 445.
Chapter 3 Ligand Electronic Properties in the Control of Redox Behavior and Reactivity toward Superoxide in Seven Coordinate Manganese Complex
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.
[17] R. H. Weiss, A. G. Flickinger, W. J. Rivers, M. M. Hardy, K. W. Aston, U. S.
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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.
4.3 Experimental Section
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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,
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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 Results and Discussion
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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)
Table 4-4 selected bond lengths (Å) and bond angles (deg) of 8
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 4 Structural Features in Control of Reactivity toward Superoxide in Manganese and Iron Complexes
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
101
5.3 Experimental Section
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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.
Synthesis of Malonate S3:
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,
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Synthesis of Malonate S4:
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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,
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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]
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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).
5.4 Results and Discussion
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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).
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Chapter 5 High Catalytic Activity of Dendritic C60 Monoadducts in Metal-Free Superoxide Dismutation
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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.
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
126
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
Design and Evaluation of Seven-coordinate Manganese and Iron Complexes, and Fullerene derivatives, as SOD mimics
128
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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