Antioxidant Activity Measured by Fluorescence - 1col

Antioxidant Activity Measured by Fluorescence:
Investigation of Antioxidant and Probe Structure as well as

their Mobility and Position
by
Roland Meyer
A thesis submitted in partial fulfillment

of the requirements for the degree of
Doctor of Philosophy
in Chemistry
Approved, Thesis Committee

Prof. Dr. Werner M. Nau, supervisor
Prof. Dr. Mathias Winterhalter, internal member
Prof. Dr. Detlef Gabel, external member
Date of defense: 13.05.2011
School of Engineering and Science
Jacobs University Bremen, Germany

To my parents

Optimisten sind Menschen,

die wissen wie schlecht die Welt ist;
Pessimisten sind Menschen,
die es tglich neu erleben mssen.
Sir Peter Ustinov
vii
Table of Contents
Acknowledgements ......................................................................................................ix
List of Publications........................................................................................................x
List of Attended Conferences........................................................................................x
List of Acronyms..........................................................................................................xi
Abstract ..................................................................................................................... xiii
Chapter 1 Introduction
1.1 Scopeofthethesis ........................................................................................................................................... 3
1.1.1 Summaryandaims................................................................................................................................. 3
1.2 Freeradicals,andtheirimportanceinbiologicalsystems............................................................. 5
1.3 Antioxidantdefensesystems:Anoverview.......................................................................................... 6
1.4 Lowmolecularweightchainbreakingantioxidantsinandassociatedwithlipids ............ 7
1.4.1 VitaminE .................................................................................................................................................... 8
1.4.2 VitaminC ..................................................................................................................................................12
1.5 Experimentalapproachestoelucidateantioxidantaction ..........................................................13
1.5.1 DBOasafluorescentprobeforantioxidants ............................................................................15
1.5.1.1 FluorazophoreL:AlipidsolubleDBOderivative ..............................................................17
1.5.1.2 SynthesisofFluorazophoreL .....................................................................................................18
1.6 DBOanditsderivativesasfluorescentprobesinthedeterminationofantioxidant
action.....................................................................................................................................................................20
1.6.1 QuenchingofFluorazophoreLbyvitaminC ............................................................................21
1.6.2 QuenchingofFluorazophoreLbyvitaminE............................................................................22
Chapter 2 Materials and Methods

2.1 Materials ............................................................................................................................................................29
2.2 Liposomepreparation..................................................................................................................................29
2.3 Fluorescencespectroscopy........................................................................................................................30
2.4 Liposomecharacterization ........................................................................................................................30
2.5 Mathematicalanalysisfordeterminationofthemutuallateraldiffusioncoefficient......31
viii
Chapter 3 Antioxidant Activity Measured by Fluorescence: Investigation

of Antioxidant and Probe Structure as well as their Mobility and Position
3.1 Analysisofasyntheticanalogueoftocopherol............................................................................ 35
3.2 EffectsofsubstitutiononDBOfluorescence...................................................................................... 39
3.3 Phasedependenceofantioxidantdiffusion ....................................................................................... 43
3.4 pHdependenceofLascorbyl6palmitateantioxidantactivity................................................ 47
3.5 Conclusions ...................................................................................................................................................... 51
3.6 Perspectives..................................................................................................................................................... 51
Chapter 4 Industry Project

4.1 Samples .............................................................................................................................................................. 57
4.2 Parameterdeterminationtechniques .................................................................................................. 57
4.3 VitaminE ........................................................................................................................................................... 58
4.4 VitaminCandUricAcid .............................................................................................................................. 58
4.5 Totalantioxidantpower[92] ....................................................................................................................... 59
4.6 Isoprostane[93]andcreatinine[94] ............................................................................................................ 59
4.7 GSH[101] ............................................................................................................................................................... 62
References ............................................................................................... 65
Chapter 5 Appendix
AppendixI:HowtoperformaglobalfittodetermineDL .......................................................................... 73
Appendix II: Article, Journal of the American Chemical Society.....................................................79
Appendix III: Article, Photochemical & Photobiological Sciences ................................................111
Appendix IV: Article, Langmuir......................................................................................................119
AppendixV:Lebenslauf..........................................................................................................................................127
ix
Acknowledgements
I would like to express my gratitude to my supervisor Prof. Dr. Werner M. Nau, who
took a wayward biologist under his wing and allowed him to delve into the
fascinating world of photochemistry. Without his ideas, capable guidance, constant
support and many fruitful discussions this thesis would not have been possible.
I would like to thank Prof. Dr. Mathias Winterhalter and Prof. Dr. Detlef Gabel for
their participation as co-referees.
I am grateful to the members of the Nau workgroup, both past and present, for a
dynamic, friendly, and enjoyable atmosphere. In particular I would like to thank Dr.
Harekrushna Sahoo for all the knowledge regarding instrumentation and
methodology he passed on to me and Thomas Schwarzlose for helping me out with
the synthetic procedures, and for supplying most of the raw materials used in my
experiments. I am grateful to Dr. Andreas Sonnen for introducing me to the global
fitting procedures and the lifetime measurements for lateral diffusion, and to Anja
Mller for working with me in the framework of the industry project. I also thank
Roy DSouza for his tireless help with the more artistic endeavors in order to provide
good-looking figures and schemes.
I am indebted to Prof. Dr. Porter who chose to include our workgroup in his analysis
of the synthetic antioxidant N-tocopherol.
I would like to thank the workgroups of Prof. Dr. Schwaneberg for their support in
measuring multiplates and in handling biological waste, and of Prof. Dr. Winterhalter
for allowing me to perform light-scattering and differential scanning calorimetry
measurements with their instruments.
Finally, I am extremely grateful to my father, without whose constant support, both

morally and financially, I would not have been able to finish.
x
List of Publications
T. Nam, C. L. Rector, H. Kim, A. F.-P. Sonnen, R. Meyer, W. M. Nau, J.
Atkinson, J. Rintoul, D. A. Pratt, N. A. Porter, Tetrahydro-1,8-naphthyridinol
Analogues of -Tocopherol as Antioxidants in Lipid Membranes and Low-Density
Lipoproteins, J. Am. Chem. Soc. 2007, 129, 10211-10219
R. Meyer, X. Zhang, W. M. Nau, Effect of bridgehead substitution on the

fluorescence quenching of 2,3-diazabicyclo[2.2.2]-oct-2-enes by solvents and
antioxidants, Photochem. Photobiol. Sci. 2009, 8, 1694-1700
R. Meyer, A. F.-P. Sonnen, W. M. Nau, Phase-Dependent Lateral Diffusion of -

Tocopherol in DPPC Liposomes Monitored by Fluorescence Quenching, Langmuir
2010, 26, 14723-14729
List of Attended Conferences

NRP Final Symposium: Supramolecular Functional Materials, 16.06. - 18.06.2005,
Murten (CH)
ChemieContact Innovation sucht Partner des VCI (Verband der Chemischen

Industrie e.V.), 10.10.2006, Hamburg (D)
4th European Short Course on "Principles and Applications of Time-Resolved

Fluorescence Spectroscopy, 30.10. - 03.11.2006, Berlin (D)
21. Lecture Conference der Fachgruppe Photochemie, 06.10. - 08.10.2008, Bielefeld

(D)
Bremen Molecular and Marine Biology Retreat, 26.01 27.01.2007, Etelsen (D)
NanoFun Center and Nanomol Graduate Retreat, 12.01 14.01.2011 Flambacher

Mhle, Clausthal-Zellerfeld (D)
4. Treffen der Norddeutschen Biophysiker, 21.01.2011, Forschungzentrum Borstel

(D)
xi
List of Acronyms
A6P L-ascorbyl-6-palmitate
AMVN 2,2-azobis-(2,4-dimethylvaleronitrile)
BDE H-bond dissociation enthalpy
DBO 2,3-diazabicyclo[2.2.2]oct-2-ene
DBO-R 1,4-dichloro-2,3-diazabicyclo[2.2.2]oct-2-ene
DBO-S 5-methyl-1-isopropyl-2,3-diazabicyclo[2.2.2]oct-2-ene
DMAP 4-dimethylaminopyridine
DNA deoxyribonucleic acid
DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
DSC differential scanning calorimetry
DTNB 5,5-dithiobis(2-nitrobenzoic acid)
EA activation energy
EDTA ethylenediaminetetraacetate
ELISA Enzyme-linked immunosorbent assay
EP electron transfer processes
EPR electron paramagnetic resonance spectroscopy
FRAP fluorescence recovery after photobleaching
GSH reduced glutathione (-glutamyl-cysteinylglycine)
GSSG glutathione disulfide
HPLC high performance liquid chromatography
hTTP human tocopherol transfer protein
IR infrared
ISC inter-system crossing
LDL low density lipoprotein
MDA malondialdehyde
MTAD 4-N-methyl-1,2,4-triazoline-3,5-dione
N-TOH naphtyridinol-based tocopherol
xii
NADPH nicotinamide adenine dinucleotide phosphate (reduced)

NDP-PE N-4-nitro-benz-2-oxa-1,3-diazole phosphatidylethanolamine
NMR nuclear magnetic resonance
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
RNS reactive nitrogen species
ROS reactive oxygen species
SCE saturated calomel electrode
SDS sodium dodecyl sulfate
TCSPC time-correlated single-photon counting
TNB 5-thio-2-nitrobenzoic acid
Triton XR-100 t-octyl-cyclohexylpolyethoxyethanol
UV ultraviolet
xiii
Abstract
This doctoral thesis is concerned with determining antioxidant mobility by
fluorescence, especially in lipid membranes. It expands on previously determined
data regarding antioxidant diffusion by employing different lipids, synthetic
antioxidants, and varying pH. The probes used are derivatives of 2,3-
diazabicyclo[2.2.2]oct-2-ene (DBO), which are known for their exceptionally long
lifetimes in aerated aqueous media (325 ns in H2O under air). Their n,* singlet-
excited state is efficiently quenched by an aborted hydrogen abstraction mechanism,
which makes them ideally suited to probe antioxidant diffusion.
During the course of my Ph. D. thesis I was able to expand the toolbox of available
DBO derivatives with a very reactive one, Fluorazophore-R, and a more acid-stable
lipophilic derivative, Fluorazophore-LE.
I was also able to determine the mutual lateral diffusion coefficient of a synthetic
vitamin E derivative with single-photon counting measurements. This N-tocopherol
displays higher mutual lateral diffusion coefficients than -tocopherol (5.4 107 cm
1
s1 vs. 3.73 107 cm1 s1 at 40C, respectively) with a very similar activation
energy of 43 6 kJ/mol (compared to 47 5 kJ/mol), which is indicative of a higher
antioxidant potency.
Expanding on previous experiments I was also able to probe the differing behavior of
-tocopherol at the phase transition threshold of liposomes composed of a high
melting fully saturated lipid, DPPC. Above the phase transition temperature, mutual
lateral diffusion coefficients of 107 cm2 s1 were found, similar to the ones in POPC,
while above the pre-transition temperature the coefficients were 108 cm2 s1. This
confirms the high accuracy of our measurement principle.
Furthermore, the first steps have been taken in investigating the pH dependence of the
synthetic antioxidant L-ascorbyl-6-palmitate, which is only a decent lipophilic
antioxidant in low pH environments.
Additionally, I have worked on a third party funded research project for a company
analyzing urine and blood samples, the results of which I am not at liberty to discuss.

Chapter 1
Introduction
Chapter 1: Introduction 3
1 Introduction
1.1 Scope of the thesis

The main focus of this thesis is the investigation of antioxidant action in lipid
systems. Lipid-soluble antioxidants are an essential part of the membrane
environment in organisms. To assess them it is imperative to have a working model
for their interactions with free radicals that is robust and can be applied to various
lipids. In particular the elucidation of diffusion phenomena, one of the two governing
factors for antioxidant potency, is not straightforward. In my Ph.D. thesis I will
outline the work already done in this regard, as well as further scrutinize the tools that
were chosen for this very purpose. The introduction will highlight the problems faced
when trying to investigate diffusion, especially in lipid systems, the importance of
low molecular weight chain-breaking antioxidants, the methods used so far to
investigate diffusion in lipid systems, and a thorough discussion of our approach,
which includes previously published results. Chapter 3 will detail the experiments
performed during my Ph.D., while Chapter 4 will outline an antioxidant-related
industry project for which I performed the experiments.
1.1.1 Summary and aims

Antioxidants are a fascinating topic with numerous real-world applications, both in
a scientific as well as an industrial sense. I first became interested in antioxidants as a
research topic when I took over the determination of the mutual lateral diffusion
coefficient of N-TOH (see Section 3.1) from a former member of the workgroup.
Antioxidants, especially lipophilic ones, albeit studied to some extent, still lack vital
basic data to actually understand what is happening in a cell membrane. One of the
problems faced by anyone intending to study antioxidants is the loose placement of
the topic in the wider context of the disciplines. While biophysicist, medical
scientists, biologists, and the industry are all highly interested in the topic as a whole,
each of the groups tends to use the methods they are accustomed to in order to gain
new insight. That means, at least for the biologists studying antioxidants, that the
model systems they choose are almost always whole cell membranes. While this is
certainly a viable context, the problems of investigating diffusion in actual biological
membranes are considerable, due to the abundance of membrane additives in nature

(which sometimes make up the majority of lipid membranes, instead of the lipids
themselves) and the many different lipids of which a membrane is composed. For me,
the most important task was to establish a reliable method by which diffusion in
lipids can be measured, in an artificial membrane model. Once these data are
available, steps can be taken to investigate more complex systems.
With DBO, a simple bicyclic azoalkane, as a fluorescent probe (see Section 1.5.1),
it is possible to measure both governing factors for lipophilic antioxidant activity, the
hydrogen donor propensity and the lateral diffusion coefficient. Only with both of
these can one assess the potency of new synthetic antioxidants. Coming back to the
N-TOH, I was able to fit the measured curves in order to conclude that this was,
indeed, a very active lipophilic antioxidant, and, in conjunction with the other
experiments performed, a compound worth investigating further. The next logical
step to take in the investigation of lateral diffusion of antioxidants was the change in
the type of lipid used to construct the liposomes. Instead of a monounsaturated lipid a
fully saturated lipid was used, which gave me the opportunity to investigate the
behavior of the antioxidant above and below the phase transition temperature. I was
very surprised that our method was sufficiently sensitive to measure the very small
lateral diffusion coefficient even in the ripple phase of our liposomes. This is very
promising, considering the differences in lipid composition of biological membranes.
Another important task to further enhance our method is the investigation of new
probes based on DBO. I could show that bridgehead substitution of DBO drastically
alters the photophysical properties of the compound, and that this toolbox approach
is very practical when measuring a large range of antioxidants, with all their diverse
constituents.
In summary I would like to emphasize that antioxidant activity studies are still in
their infancy, and that, if one ever hopes to fully understand the processes they are
involved in, a lot more basic research must be performed. As a start, it would help to
construct a table containing all of the biologically relevant lipids and the diffusion
coefficients of lipid-soluble antioxidants therein. Without such a table, the
investigation of synthetic antioxidants will not be feasible. For this, new probes will
have to be designed and synthesized, but the method, which I have helped to develop
in this thesis, is so robust that it is worth putting more effort into it.
1.2 Free radicals, and their importance in biological systems

Free radicals have been revealed as a source of many human ailments. A radical
has been defined as a molecular species that contains an unpaired electron in an
atomic orbital and is capable of independent existence.[1] The single unpaired electron
that is the defining characteristic for radicals leads to a very high, aggressive
reactivity. The origin of radicals in biological systems is manifold, they may stem
from environmental radiation or chemical accidents during normal cell function.
These endogenous sources include mitochondrial leak, respiratory burst, enzyme
reactions, and autoxidation reactions while environmental sources like cigarette
smoke, pollutants, UV- and ionizing- radiation and xenobiotics are known to partake
in radical generation.[2] One of the primary radicals generated is OH, but due to its
high reactivity and short half-life, most of the investigation into radical-damage in
vivo is directed towards the downstream processes.
The most common reactive oxygen species (ROS) and reactive nitrogen species
(RNS) encountered are superoxide, hydroxyl, peroxyl, alkoxyl, hydroperoxyl, nitric-
oxide and nitrogen dioxide radicals. Hydrogen peroxide, although not a radical itself,
is known to be a very important progenitor for radicals and as such is worth
mentioning. Transition metals, facilitators of free radical generation as well, will only
be mentioned briefly.
Why do radicals receive so much attention? Their reactivity enables them to

interact with almost any molecule in their vicinity, and they can seriously alter
biological molecules, even to an extent where these cease to function or even become
disruptive to the organism. In addition to beneficial applications, for example during
phagocytosis,[3] DNA, proteins[4] and lipids,[5] the most important classes of
molecules in biological systems, are the most interesting to study with regard to
radical damage. Adding the ubiquity of free radicals or free radical generating agents
in nature makes for a very interesting field of study.
As life evolved from basic unicellular bacteria to the conglomerates of millions of

cells that make up all of the higher life forms, drastic changes in the environment
occurred. The continual rise of atmospheric O2 levels, starting approximately 2.3
billion years ago and reaching contents of 1% around 1.3 billon years ago,[6] to the
present-day 21%, posed a new threat to the organic life that had, thus far, mostly
consisted of anaerobic bacteria.[7] Many of these bacteria are thought to have died out
in conjunction with the rising O2 levels, while others developed strategies to cope
with the new situation.[8] The most primitive of these strategies was to avoid oxygen
altogether, which meant moving to habitats where oxygen could not penetrate, for
example sediments. Many of these anaerobic bacteria and their descendents still exist
today, and some have even developed methods that allow them to use their motility to
stay in habitats most favorable for their proliferation.[9] Other species, especially
those that changed their biochemistry to use oxygen in their respiratory processes,
adapted to the higher oxidative stress by developing various countermeasures to not
only survive, but thrive in environments with high partial oxygen pressure.[4]
1.3 Antioxidant defense systems: An overview

Halliwell and Gutteridge introduced a broad and very useful definition of the term
antioxidant: Any substance that, when present at low concentrations when compared
with those of an oxidisable substrate, significantly delays or prevents oxidation of
that substrate.[1] An oxidisable substrate in this definition includes every molecule
found in vivo. Four big classes of antioxidants are recognized:
(1) Pro-oxidant restricting compounds, which minimize the availability of

certain ions or molecules like iron, copper or haem, including transferrin,
haptoglobin, caeruloplasmin, and ferritin.
(2) Proteins that protect biomolecules by other mechanisms, for example
heat shock proteins which help retain structural motifs of proteins.
(3) Catalysts, mostly enzymes, which remove free radicals as well as other
reactive species, including such enzymes as superoxide dismutase, catalase,
and peroxidase.
(4) Low molecular mass compounds that scavenge reactive oxygen or

nitrogen species. These include the vitamin E constituents, ascorbic acid, uric
acid, glutathione, and numerous others.
A fifth class is sometimes added to this list, the repair enzymes, which help
minimize the effect of free radicals on cells and tissue.[1] These actually do not adhere
to the definition of antioxidant given above, since they do not prevent or even delay
damage. It is therefore not desirable to weaken the definition by their inclusion.
Of these four classes only the last one will be investigated in the scope of this
Ph.D. work. Special emphasis will be put on vitamin E, a class of lipid-soluble
antioxidants, and on ascorbic acid (vitamin C), due to its close in vivo connection to
vitamin E and radicals in membranes. Several other antioxidants will be mentioned
briefly in their respective chapters.
1.4 Low molecular weight chain-breaking antioxidants in and

associated with lipids
Radical deactivation by low molecular weight chain-breaking antioxidants is based
on the donation of a hydrogen atom. In essence the antioxidant becomes a radical
itself, but one that is much less reactive than the lipid-radical. This new radical is
recycled by various means, for example the -tocopheroxyl radical can be
regenerated by ascorbate, or can undergo further oxidation to tocopherylquinone.[1]
Two major factors determine the potency of any low molecular weight chain-
breaking antioxidant: the hydrogen atom donor propensity, and the diffusion
coefficient. While the former is based on the molecular structure of the antioxidant
molecule[10] with extraneous factors contributing only to a small degree, the diffusion
coefficient is extremely dependent on the environment. In aqueous solution most of
the radical scavenging processes are close to diffusion controlled, but for antioxidants
incorporated into the lipid membrane, for example vitamin E, the situation is less
straightforward. The membrane composition, i.e., which lipid or lipid mixtures
constitute the membrane as well as the amount and type of additives, e.g. proteins and
compounds like cholesterol, define the boundary conditions for diffusion.
One of the fundamental features that distinguishes living organism from inanimate
matter is the ability to differentiate itself from its surroundings. Without a working
compartmentalization many important processes are next to unthinkable, such as
energy generation, selective signaling, substance uptake and protection against
xenobiotics or bacteria. Lipid-soluble low molecular weight chain-breaking
antioxidants are so interesting because they are located in this cellular organelle,
which is not easily penetrated from the outside. In medicine, lipid-soluble
antioxidants receive special attention due to the importance of the preventive effects
they have for certain human ailments.[11] For example, the peroxidation of lipids in
biomembranes[12] and of low density lipoprotein (LDL) particles[13] may be one of the
factors contributing to atherosclerosis. Low-density lipoprotein (LDL) particles, the
major carrier of cholesterol in blood, form fatty streaks[14] on the interior walls of
arteries. From an anthropogenic point of view the vitamin E constituents are
especially noteworthy as the most important, albeit not the only, lipid-soluble
antioxidants in mammals.
1.4.1 Vitamin E
Vitamin E is a collective term for eight different substances, which are divided
into two subclasses, the tocopherols and the tocotrienols. The name tocopherol was
coined when researchers discovered a factor important for rat reproduction,[15] from
the Greek tokos for childbirth and phero for to bring forth, with the suffix ol
added to indicate its phenolic nature. All of the vitamin E constituents contain two
structural motifs: a chromanol head (with a heterocyclic and a phenolic ring) and a
phytyl tail which contains three isolated double bonds in tocotrienols (hence their
name). Vitamin E uptake occurs via food, and dietary sources for vitamin E include
wheat-germ, vegetable oils, margarines, nuts, grains, and green leafy vegetables.[1]
Tocopherols and tocotrienols exist in four different forms each (-, -, -, -),
which differ in the methylation pattern on the chromanol-head (see Scheme 1.1).[16]
Scheme 1.1: Structure of the vitamin E constituents
R1
OH 5
7 2
R2 O R3
Vitamin E constituent R1 R2 R3 (isoprenoid tail)
!-Tocopherol CH3 CH3

"- CH3 H
#- H CH3 4' 8' 12'
$- H H
(hexahydrofarnesyl or "phytyl")
!-Tocotrienol CH3 CH3
"- CH3 H
#- H CH3
3' 7' 11'
$- H H
(farnesyl)
!!
The quantification of vitamin E constituents in vivo is not trivial. Although
literature references about their concentration in blood plasma are abundant, the
amount of tocopherols and tocotrienols in the membrane is harder to determine. Of
the many different units these values are reported in, the mole amount of vitamin E
per mole of oxidizable lipid is one of the most useful. For example, the mole
percentage of -tocopherol per mole phospholipid in human platelets was reported to
be 0.3% mol.[17] For rat liver microsomes a value of 1% mol was recovered,[18] and
overall the concentration in tissue determined to date seems to be in the range of
0.1% mol to 1% mol, which corresponds to approximately 1 molecule of -
tocopherol per 1000 phospholipid-molecules. A similar value was also recovered in a
more recent determination for human platelets with a ratio of 220:1.[19]
-Tocopherol is the most widely studied constituent of vitamin E, not only due to
it being regarded as the most active,[20] but also because of the, consequently, best
commercial availability. Financial restraints are also the reason that the all-racemic
-tocopherol steroisomer is used for many experimental investigations. While the
RRR-steroisomer is the naturally occurring one, the all-rac form can be used for
experiments focusing on simple liposome-based membrane models. For example,
lateral diffusion does not change between stereoisomers.[21] When going to more
complex, and, thus, more biologically relevant, membrane assemblies with multiple
lipids and proteins, care has to be taken when employing the all-rac -tocopherol.
Studies with cholesterol in membranes have shown a selective binding of the
different cholesterol-steroisomers to lipids and proteins,[22] and if -tocopherol
behaves in a similar fashion, the all-rac form might not correctly resemble the in vivo
situation. For cell studies it is also imperative to consider that only -tocopherol
meets the dietary requirements for humans, all others are only poorly recognized by
the hepatic -tocopherol transfer protein. Even supplementing the diet of an animal
lacking this transporter with high amounts of other vitamin E constituents has been
shown to cause severe cases of vitamin E deficiency.[23]
The exact position that -tocopherol occupies in a membrane environment is not

easy to pinpoint. The fact that -tocopherol intercalates in lipids is uncontested, but
the horizontal positioning, especially with regard to the penetration depth of the
headgroup, i.e., how far away it is from the lipid-water interface region has been
subject to extensive research. Three different models have been proposed by
Fukuzawa et. al.[24] Model A puts the chromal headgroup close to the interfacial
region, accessible to ascorbate from the aqueous phase. Model B has the headgroup
slightly depressed, where it may interact with phosphate oxygen or acyl-ester oxygen
atoms, depending on lipid type. The last model has the molecule deeply submerged in
the bilayer, closer to the unsaturated sites that would generate peroxyl-radicals during
polyunsaturated fatty acid oxidation.[24] Further investigations, for example by
elucidating the ability to spare different doxyl-stearates from 2,2-azobis-(2,4-
dimethylvaleronitrile) (AMVN) radical-mediated destruction,[25] or by nuclear
magnetic resonance (NMR) chemical shift-polarity correlation, reveal that -
tocopherol does not occupy a single environment but is located between Model B and
C, and which enables it to interact with aqueous molecules (see Scheme 1.2).[26]
Scheme 1.2: Location of -tocopherol in a membrane: a) near the interfacial region,

b) slightly depressed, possibly hydrogen bonded to either phosphate
oxygen or acyl-ester oxygen atoms, and c) deeply submerged, closer to
unsaturated sites[24, 26-27]
a) b) c)
N N N
O O HO O O O O
P P P
O O O O O O
O O O O HO O O
O O O O
O O O
HO
The physiological function of vitamin E is still under debate. A long known and
important function is the inhibition of lipid peroxidation by scavenging chain-
propagating lipid peroxyl radicals.[28] The chain reaction[12b] initiated by lipid
peroxidation can ultimately lead to the rupturing of the cell, or at least to damage of
the intrinsic membrane proteins. Other possible functions include, but are not limited
to, steric effects on membrane conformation, association with specific proteins or
lipids or even as regulators for protein activity or gene expression.[27, 29]
Even
prooxidant effects have been described in certain situations.[13c, 30] In a normal cellular
membrane environment the antioxidant effects of the vitamin E constituents are,
however, unquestioned. As indicated above, this is accomplished by the donation of
the hydrogen atom bound weakly to the phenolic hydroxyl group in the chromanol
headgroup.[31] Its accessibility from within the membrane as well as from the aqueous
bulk leads to its high antioxidant potency. The accessibility of the radical to the
antioxidant is explained by the floating-radical theory, which is based on the
polarity of the radical, as visualized in Scheme 1.3.[32]
Scheme 1.3: Radical scavenging by antioxidants according to the floating radical

theory
HO HO HO
O O O
+O2 +O2
O OH O
Chain propagation
O O O
Radical "floats"
toward the polar
headgroups
O Radical deactivation
Lateral diffusion of HOCH2 O
HO by ascorbate
H O OH
!-tocopherol
O O O
O HO O HO O HO
O O O
Major Minor
pathway pathway
"Deactivation" of Regeneration of the !-tocopheroxyl

the radical by radical by ascorbate
hyrogen-atom
donation Regeneration of ascorbate
O O O
O CH2OH O CH2OH e / H O CH2OH
HO OH O OH HO OH
O H O H O H
OH
O O HO
O O
OH
O

1.4.2 Vitamin C
Ascorbic acid, also known as vitamin C, is a chain-breaking low molecular weight
antioxidant as well, but, in contrast to vitamin E, it is water-soluble. Plants and most
animals are able to synthesize ascorbic acid from glucose, but humans and several
other animals have lost the terminal enzyme necessary during evolution, and
conversely have to acquire it from their diet. Apart from its function as an antioxidant
ascorbic acid is also a cofactor for different enzymes, for example, the ones involved
in collagen synthesis. A lack of vitamin C leads to scurvy, a well known scourge of

the early day sailors.[1]
In addition to its antioxidant action in solution and its interaction with

membranous radicals, ascorbic acid can also regenerate -tocopheroxyl radicals in
the membrane (see Scheme 1.3). [13a, 33] Due to its first pKa at 4.04[34] ascorbic acid is
mostly found in its monoanionic form at neutral pH (ascorbate). As many
applications depend on a lower pH, for example in canned beverages, preserved fruits
or salad dressings, the less antioxidatively active protonated form (HA2)[13a, 31b, 33-35] is
predominant. A synthetic form of ascorbic acid, L-ascorbyl-6-palmitate, is of
importance as a cheaper alternative to vitamin E in industry (see Scheme 1.4).[36]
Scheme 1.4: Ascorbic acid at different pH and L-ascorbyl-6-palmitate
OH OH OH OH
O O
O C O C
H H2 H H2
HO O HO OH
Ascorbic acid at physiological pH Ascorbic acid below pH 4
OH
O O
O
O
HO O
Ascorbyl-6-palmitate at physiological pH
1.5 Experimental approaches to elucidate antioxidant action

A plethora of different experimental approaches, such as IR and Raman
spectroscopy,[37] electron paramagnetic resonance spectroscopy (EPR also referred to
as ESR (electron spin resonance)) with nitroxides as spin probes,[25] NMR
techniques,[12b] and intrinsic -Toc fluorescence measurements[38] have been
conducted to elucidate antioxidant activity in lipid systems, and to further enhance the
understanding of lipid systems themselves. A short, but by no means comprehensive,
account of the most relevant techniques and selected results shall be attempted in the
following paragraphs.
EPR is used in the investigation of radicals and their chemical reactions.

Interesting insights gained via this technique include the higher membrane
disorganization for -tocopherol when compared to -tocotrienol (which does not
seem to increase diffusion, see below),[39] tocopherol-mediated peroxidation in LDL
particles (these prooxidant effects of antioxidants are still not fully understood), the
influence of hydrogen bond donors on the stability of phenoxyl radicals,[40] or the
recycling of vitamin E by lipid-insoluble antioxidants, for example ascorbate, or
enzymes like cytochrome b5-reductase.[41]
NMR has, by contrast, mostly been employed in the elucidation of lipid lateral
diffusion, and the influence of additives such as cholesterol thereon,[42] a
phenomenon of high impact to the physiological role of the vitamin E constituents.
Other areas in which NMR studies have proven to be invaluable are the determination
of lipid domains (so-called rafts) as well as the corresponding process of demixing,
chiral effects[43] and the influence of solid-like obstacles, for example proteins, on
lipid diffusion.[44]
Fluorescence recovery after photobleaching (FRAP) employs fluorescently labeled

probes to investigated lipid dynamics. However, labels such as like N-4-nitro-benz-2-
oxa-1,3-diazole phosphatidylethanolamine (NDP-PE) have the drawback that they,
due to their bulkiness, change the system considerably. Nonetheless, the lipid
diffusion coefficients studied by FRAP with[45] or without cholesterol[46] are very
similar to those garnered by other methods, e.g NMR.[47] While improved FRAP
techniques have been developed[48] the overall impact of FRAP has lessened to a
supportive role and made room for ESR- and NMR-based experiments.
The measurement of the actual lipid peroxidation has been, and still is, one of the
most common methods to evaluate oxidative stress and antioxidant activity. Lipid
peroxidation is monitored by reporter compounds, such as thiobarbituric acid-reactive
substances (malondialdehyde, MDA), or by investigating the pressure of oxygen. The
conundrum regarding the in vivo antioxidant potency of -tocopherol vs. its reported
prooxidant effects has been investigated by lipid peroxidation studies,[49] as has the
influence of carotenoids on lipid peroxidation in liposomes.[50] The model for -
tocopherol and ascorbic acid synergism is also largely based on lipid peroxidation
studies,[51] as are experiments regarding mice lacking a crucial tocopherol transporter
protein.[52] Newer research, especially in the field of food chemistry has also gainfully
employed this technique, for example to assess antioxidative compounds from the
outer scales of onions[53] or to compare fruit juices with regard to their antioxidant
capacity and influence on fruit fly development.[54]
Lastly, several methods most commonly employed in the field of medicine are
simply physiological tests that scan for lipid peroxidation by analyzing byproducts.
These tests have in common that they constitute a fast and non-invasive way to
measure lipid peroxidation, especially in clinical trials. Two of the most widely used
biomarkers are the exhalation of pentane (sometimes ethane as well)[55] and the
amount of isoprostane in urine.[56]
Closely related to antioxidant research is the investigation of the influence of

cholesterol on membranes, which is one of natures most common membrane
additives, and might even be an adaptation of cells to high oxygen partial pressure.[57]
The influence of the phase-ordering in binary lipid mixtures containing cholesterol
has been investigated,[58] but the influence on antioxidant behavior has, so far, not
been in the focus of research, and neither has the related phenomenon of phase
transition and its impact.
1.5.1 DBO as a fluorescent probe for antioxidants

The above-mentioned methods, while suitable for their respective research topic,
fall short in regard to probing the lateral diffusion coefficient in an environment
mimicking the situation in biological membranes as closely as possible. Most of the
fluorescent probes employed with these techniques are bulky and do not resemble
lipid peroxyl radicals morphologically to a very high degree, e.g. NDP-PE, or the
measurement technique is not suitable as a whole, e.g. NMR. A new kind of
molecular probe for antioxidant activity was envisioned by Nau and coworkers, based
on the fluorescent diaza compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO).[31b] DBO
has a number of characteristics that make it very well suited for fluorescence-based
research. DBO is uncharged, small, nearly spherical and has the longest known
fluorescence lifetime in aerated solvents (325 ns in H2O). Substitution, primarily at
the bridgehead carbon, is possible and has yielded an ever-growing family of

derivatives (see Scheme 1.5). [59]
Scheme 1.5: The fluorazophore dye family.

N N N N N
N N N N Cl N
CH2NH2 Cl
O Me O O
O FluorazophoreL FluorazophoreA FluorazophoreR
Si
Ph Me
N FluorazophoreA250
N
N
N
O N
N
O
O
* O FluorazophoreP NC(H3C)2
P
N(iPr)2
O Fluorazophore-amidite
N
FluorazophoreC N
O
S
N N N
O HN
N N N
O
FmocHN COOH
"caged" CH2OH
FluorazophoreS Fluorazophore FluorazophoreH Fmoc-Fluorazophore FluorazophoreLE
The n,* singlet-excited state of DBO can be efficiently quenched by substances

with a high hydrogen donor propensity, similar to the deactivation of radicals (which
DBO resembles in regard to its lone energetically elevated electron), and so is ideally
suited for antioxidant experiments.[60] The quencher molecule is weakly coordinated
to the azo group, but the reaction is not completed on the exited state energy surface.
This mechanism is called aborted hydrogen atom abstraction.[61] The excited state
(S1) and ground state (S0) potential energy surfaces cross in a conical intersection,
before the C-H bond is broken and the N-H bond is formed, in an efficient
radiationless decay channel.[62] The early experiments established DBO as a novel
way to monitor antioxidant behavior in solution, with the major advantages of the
high resolution inherent to fluorescence techniques and the high photostability of
DBO (i.e., the probe is not consumed in the experiment), which makes the elucidation
of kinetics relatively easy and straightforward. Although the solvent effects on DBO
fluorescence are quite severe[63] it has been shown that a derivative of DBO, dubbed
Fluorazophore-S, exhibits a higher selectivity for antioxidants at the cost of lower
reactivity.[64] In addition to the assessment of the antioxidant capabilities of vitamin E
and C, discussed below, other antioxidants, for example melanin and its precursors,
have also been successfully analyzed with DBO as a probe.[65]
1.5.1.1 Fluorazophore-L: A lipid-soluble DBO derivative

The next step to be taken was the synthesis and characterization of a DBO
derivative that could be employed inside the membrane to monitor the behavior of
lipi-soluble antioxidants. This so-called Fluorazophore-L, the palmitic acid derivative
of DBO, exhibits a lifetime similar to that of the parent compound (up to 325 ns in
aerated hexane).[59] Fluorazophore-L is effectively incorporated into 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC) membranes (see Scheme 1.6), and
retains a lifetime of 125 ns in this system at 25 C, which allows the observation of
quenching processes in membranes that occur in a timeframe of 108 to 105 s,[66]
which can then be used to calculated diffusion.
Scheme 1.6: Integration of Fluorazophore-L and -tocopherol in a lipid membrane

leaflet
N
HO
N
O O O O O O O O O O O O
O O O O O O O O O O O O
O
O O
1.5.1.2 Synthesis of Fluorazophore-L

The lipid containing derivative of DBO, Fluorazophore-L, was synthesized
starting with Fluorazophore-H (8 in Scheme 1.7), which contains a hydroxymethyl
group in a bridgehead position. This allows rapid synthetic functionalizations with
different groups. This precursor is formed by the synthetic procedure outlined below
(see Scheme 1.7).
In step a) crotonaldehyde (1) reacts in a one-pot reaction with diethylamine in a

combined addition to the C=C double-bond. The following hemiaminal formation
succeeded by a thermal elimination results in the formation of the unstable diene (2).
The Diels-Alder reaction with acrolein in diethylether forms the cyclic adduct (3)
which readily looses diethylamine hydrochloride to form the cyclic diene aldehyde
(4). Reduction with sodiumborohydride produces the corresponding allylic alcohol
(5). The subsequent reaction with the strong aza-dienophile 4-N-methyl-1,2,4-
triazolin-3,5-dione (MTAD) results in the formation of a polycyclic ring system (6).
After catalytic hydrogenation to (7) and subsequent basic hydrolysis with alcoholic
potassium hydroxide while stirring under air for several hours, this forms the desired
product (8) with an overall respectable yield (ca. 24%).[67]
Fatty acids can be converted to their ester derivatives by activating the carboxylic
acid functionality, e.g. as an acid chloride (see Scheme 1.8).[68] Since palmitoyl
chloride is commercially available, it was used as the reagent of choice to prepare the
Fluorazophore-H ester compound. The acid chloride reacts with the hydroxy group of
the alcohol to form the ester and hydrogen chloride. The formed hydrogen chloride is
trapped by addition of the base triethylamine, which pulls the equilibrium of this
reversible reaction to the side of the ester. That causes the precipitation of its
hydrochloride salt, which, at the end, is simply filtered off. The addition of a catalytic
amount of 4-dimethylaminopyridine further improves the reaction rate and the final
yield of the ester compound (approx. 96%).[69]
Scheme 1.7: Synthesis of the Fluorazophore-H
NEt2
O H NEt2
1) K2CO3, 2 HNEt2 CH2CHCHO, Et2O CHO
2) !, HNEt2 0 C, 5 h
1 2 3
CHO
conc. HCl NaBH4, MeOH OH
r.t., 90 min 0 C, 18 h
4 5
O O
MTAD, CH2Cl2 N H2, Pd/C N
0 C N N CH3 EtOH N N CH3
OH O OH O
6 7
1) i-PrOH/KOH
N
2) !, 15 h, O2
N
OH
8
Scheme 1.8: Synthesis of Fluorazphore-L, 1-hydroxymethyl-DBO-palmitoyl ester
N
N
OH
Fluorazophore-H
Cl
O palmitoyl chloride
CH2Cl2 / NEt3 / DMAP
N 0C to r.t. / 18h
N
O
O
1-hydroxymehtyl-DBO-palmitoyl ester
(Fluorazophore-L)
1.6 DBO and its derivatives as fluorescent probes in the

determination of antioxidant action
Incorporation of Fluorazophore-L in lipid-like assemblies has been conducted in
different ways and with different goals in mind. The particles most suited for
investigation are micelles and liposomes. The fundamental difference between them
is that liposomes possess a double membrane whereas micelles do not. This makes
liposomes the better model for biological membranes, while micelles more closely
resemble LDL particles, which contain unpolar components in their core and are
involved in the transport of cholesterol, for example.[1] A schematic representation of
radical mimicry of Fluorazophore-L in liposomes and its interaction with both
vitamin E and vitamin C can be seen in Scheme 1.9.
Scheme 1.9: Fluorazophore-L quenching by antioxidants in a liposome.
O
HOCH2 O
HO
H O OH
N * HO *
N
N N HO
N N
O O O
O O O O O
h! h!
Minor pathway Major pathway
kback DL
Fast process Lateral diffusion
DL
N H O N *HO
N N
O O
O O O O
kq
1.6.1 Quenching of Fluorazophore-L by vitamin C

Ascorbic acid, one of the most important water-soluble antioxidants in nature, is
also able to interact with radicals, or the radical mimic Fluorazophore-L, if they are
embedded in a lipid assembly. The study proving this interaction[70] yielded valuable
insight into the action of ascorbic acid in two pH regions. Vitamin C exists as a
monoanion (HA) at pH 7 and fully protonated (H2A) at pH 2.7. Vitamin C quenches
the parent compound Fluorazophore-P with a quenching rate constant of 205 x 107
M1 s1 for the HA and 120 x 107 M1 s1 for the H2A in water. If Fluorazophore-L is
incorporated in lipids, however, the trend is diametrically opposed. For example, in 1-
palmitoyl-2-oleoyl-sn-glycero-3-phophocholine (POPC) liposomes, the quenching
rate constants for HA and H2A are 1.3 and 10 x 107 M1 s1, respectively. This has
been rationalized by the relative hydrophobicity of the two forms of ascorbic acid. In
the fully protonated form, ascorbic acid forms a protective sheath around the
liposome, mediated by hydrophobic interactions between the vitamin C and the
liposome surface, and as such yields a higher local concentration. The plausibility of
this model has been increased by measurements with negatively charged sodium
dodecyl sulfate (SDS) micelles, which show a 20-times higher quenching rate
constant for H2A than for HA, which is due to the repulsion effect of the monoanion
and the charged micelle surface. This study has opened new venues of explaining
polar antioxidant action, which is of high importance for food processing and
conservation. Many applications in this field require low aqueous pH or dry
conditions, and it cannot be generally said that under these conditions the antioxidant
action of vitamin C is diminished. In a similar manner, the contributions of ascorbic
acid to the function of the aqueous humour, the fluid located in the anterior chamber
directly behind the cornea of the eye, have been investigated as well. It resembles
blood plasma in composition, but contains much less protein, less glucose, more
lactic acid, and significantly more ascorbic acid. It could be shown that the ascorbic
acid is responsible for 75 % and 85 % of the antioxidant action in porcine and bovine
aqueous humour, respectively,[71] protecting the eye from the adverse effects of
intense radiation.
1.6.2 Quenching of Fluorazophore-L by vitamin E

The same lipid systems have also been employed in the investigation of -
tocopherol, the most active antioxidant of the vitamin E constituents in solution.[21] In
contrast to the vitamin C experiments, all components are located inside the
membrane. Different lipid assemblies allow different modes of diffusion, which is
important for antioxidant action. The quenching rate constant measured in solution
only yields an estimate of how high the hydrogen donor propensity is, i.e., how
readily and quickly the radical, or radical mimic, can abstract the hydrogen atom from
the antioxidant, but the quenching capabilities of a lipophilic antioxidant are
governed mostly by the velocity at which the antioxidant can traverse the surrounding
moiety, i.e., its lateral diffusion coefficient.
In solution, molecular motion occurs at the rate of diffusion, but in the more rigid
lipid environment diffusion is markedly decreased. The different lipid-soluble
antioxidants display different diffusion according to the shape and size of the
membrane into which they are integrated. SDS micelles, t-octyl-
cyclohexylpolyethoxyethanol (Triton XR-100) micelles and POPC liposomes have to
be treated in a different fashion when mathematically describing the diffusion
processes in their membranes. SDS micelles, with a diameter of 2 nm, can be treated
according to the ideal micellar quenching model with a Poissonian statistical
distribution of quencher among the micelles.[31a, 72] The unimolecular quenching rate
constant was found to be 2.4 0.4 x 107 s1 at 27 C .[31a] For the similarly built but
larger (4 nm in diameter) Triton micelles the ideal micellar quenching model is no
longer applicable.[73] The two possible diffusion models are the free three-
dimensional diffusion and the lateral, or two-dimensional, diffusion. Lateral diffusion
describes processes where molecules move in a 2D layer, for example on the surface
of a sphere. It has been inferred that the 2D diffusion model is better able to describe
the experimental data (refer to Section 2.5 for a detailed description of the fitting
procedure). The mutual lateral diffusion (DL) coefficient describes the movement of
the probe Fluorazophore-L and its quencher -tocopherol in the confines of the lipid
layer, and is 3.5 0.1 x 107 cm2 s1 at 27 C for Triton XR-100 micelles.[31a] POPC
liposomes are not only much larger (70 nm in diameter) but also consist of a double
layer. This makes them the investigated lipid ensemble that most closely resembles
actual biomembranes. POPC is a monounsaturated phospholipid with a fatty acid
chain length of 16:0 (palmitoyl) and 18:1 (oleoyl), and a phase transition temperature
of 2 C. The treatment of the fluorescence decay traces with a global fitting
procedure allows the determination of DL, which is at 1.8 0.1 107 cm2 s1 at 27
C.[31a] With this result, Fluorazophore-L could be established as a sensitive and
relatively simple probe for antioxidant activity in membrane model systems (see also
Figure 1.1).[31a] The diffusion coefficients recovered by this method are mutual lateral
diffusion coefficients, i.e., the movement of probe and quencher, but due to the close
structural resemblance of Fluorazophore-L and -tocopherol it can be assumed that
both contribute by approximately 50%. Also of note is the high temperature
dependence of -tocopherol diffusion, and thus its high impact on the quenching rate.
Figure 1.1: Diffusion in membrane models a) in an SDS-micelle (ideal micellar

quenching) b) in a POPC liposome c) in a Triton XR-100 micelle according to lateral
(2D) diffusion d) in a Triton XR-100 micelle according to 3D-diffusion; the blue
color indicates Fluorazophore-L and the red color -tocopherol
As stated above, vitamin E is a collective term for a class of lipid-soluble

antioxidants. Experiments have been conducted to investigate the differences in
lateral diffusion of the tocopherols and tocotrienols. The quenching rate constant of
-tocopherol is roughly double that of -tocopherol in solution (3.0 x 109 M1 s1 and
1.3 x 109 M1 s1 for all-racemic mixtures, respectively). If integrated into POPC
liposomes, though, both vitamin E constituents display almost the same mutual lateral
diffusion coefficient with virtually no differences between tocopherols and
tocotrienols (1.54 x 107 cm2 s1 for -tocopherol, 1.58 x 107 cm2 s1 for -
tocotrienol, 1.48 x 107 cm2 s1 for -tocopherol and 1.53 x 107 cm2 s1 for -
tocotrienol at 25 C), although the difference gets more pronounced at higher
temperatures. This is an indication that, for reactive radicals, neither ring methylation
nor isoprenoid tail saturation is an important criterion. In case of less reactive
radicals, the methylation pattern of the chromanol group should play a role. If the
radical is not highly reactive, the antioxidant could diffuse away from the encounter
complex again before hydrogen atom transfer occurs. A high hydrogen donor
propensity will ensure hydrogen atom abstraction, thus leading to a higher antioxidant
activity.
The effect of temperature is a very pronounced one. At 40 C, the DL of -

tocopherol is 3.73 x 107 cm2 s1, more than two times higher than at 25 C. This
illustrates that temperature has a much higher impact on antioxidant diffusion than
cholesterol, a very common additive to membranes in nature. 5 mol % of cholesterol
alter the DL only insignificantly, while higher amounts (15 mol % or 30 mol %)
decrease it considerably. One must take into account, however, that the action of
cholesterol in membranes has not yet been fully understood. At higher concentrations
it seems to obstruct the path of the molecules through the bilayer, thereby hindering
diffusion. By using the DL values at different temperatures it has been possible to
calculate the activation energy (EA) which again illustrates the similarities of -
tocopherol and -tocopherol. EA for -tocopherol is 47 5 and 39 4 kJ mol1 for -
tocopherol.
The functionally familiar L-ascorbyl-6-palmitate has also been investigated

according to its lateral diffusion (see also Section 3.4). While very similar in its
quenching rate constant in water to -tocopherol 1.4 x 109 M1 s1, its lateral diffusion
coefficient is one order of magnitude smaller (0.06 x 107 cm2 s1 at 25C). The
calculated EA of 49 5 kJ mol1 is again in accordance with the values for the
vitamin E constituents. This finding can be explained by the charge carried by the
headgroup of L-ascorbyl-6-palmitate. In contrast to the uncharged chromanol group
the negative charge effectively displaces L-ascorbyl-6-palmitate from the membrane,
thus leading to an unfavorable encounter complex, requiring an up-down transversal
movement. This is similar to the quenching of Fluorazophore-L by vitamin C, which
is even farther dislocated from the membrane.[21]
Chapter 2
Materials and Methods
Chapter 2: Materials and Methods 29
2 Materials and Methods

The materials used and the methods employed throughout the experimental work
done for this thesis will be explained below to allow for easier referencing and to
prevent unnecessary repetition.
2.1 Materials
-Tocopherol was used in its all-rac form (99.6%, donation from DSM
Nutritional Products, Kaiseraugst, Switzerland). L-ascorbyl-6-palmitate was
purchased from Sigma (Seelze, Germany) and recrystallized from hexane prior to
use. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-Dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC) were obtained from Sigma and used as
delivered. All solvents were of spectroscopic grade purity; all other chemicals were
p.a. purity. The water used was always of ultra-pure grade.
2.2 Liposome preparation

All liposomes were prepared according to the ethanol injection method[74] to
allow for a homogenous distribution of -Toc and the fluorazophore in the lipid
double-layer of the liposome. Stock solutions of -Toc, Fluorazophore-L and lipid
(POPC or DPPC) were freshly prepared for each set of experiments and stored at 20
C in dark-brown graduated cylinders. To 84 L of ethanolic stock solutions of lipid
and Fluorazophore-L, at concentrations of 30 mM and 3 mM, respectively, 16 L of
ethanol containing varying amounts of -Toc were added. 84 L of this mixture were
injected into 3 mL of 0.1% NaCl solution, for which ultra-pure water was used,
yielding a liposome dispersion with a final lipid concentration of 0.7 mM and an
overall ethanol concentration of 2.2%. It was important that the aqueous solution was
vigorously stirred and heated above the main phase transition for DPPC, 50 C in our
experiments, to ensure a homogeneous distribution of the different components as
well as the formation of unilamellar liposomes of uniform size.[66]

2.3 Fluorescence spectroscopy

The fluorescence decay traces were recorded with a time-correlated single-
photon counting (TCSPC) fluorimeter (FLS920, Edinburgh Instruments Ltd.)
equipped with a PicoQuant diode laser LDH-P-CA375 (exc = 373 nm, obs = 450 nm,
full width at half maximum ca. 50 ps) for excitation. A circulation water bath (Julabo
F25/HD thermostat) was used in conjunction with the EasyTemp software package to
ensure constant temperature (0.1 C), measured with an internal reference. The
pulse frequency of the laser in the TCSPC experiments was always kept below 2% of
the inverse lifetime while the count rate has been kept at approximately 1% of the
pulse rate. The stop condition chosen was 40 min to ensure maximum comparability
between the individual measurements. The experiments have been carried out with
freshly prepared samples in quartz cuvettes, and an identically prepared reference
sample, containing neither probe nor quencher, but the same concentration of ethanol,
was measured for each temperature to correct for scattered light and instrumental
dark counts. The fluorescence lifetimes 0 required for fitting were directly analyzed
with the instrumental software by fitting monoexponential or biexponential decay
functions, where appropriate, to the decay traces. The traces in the presence of -Toc
were used directly in the global analysis to extract the diffusion coefficients with the
software ProFit.[75]
2.4 Liposome characterization

The liposome size measurements were performed by dynamic light scattering
with a Zetasizer Nano ZS 90 (Malvern Instruments), which employs a He-Ne laser at
a 90 angle (40 mW, = 633 nm). The phase transitions were also followed by
differential scanning calorimetry (DSC) on a VP-DSC Instrument from MicroCal
(MA). The employed samples contained exactly the same amount of lipid, quencher,
and probe as those measured by TCSPC.
2.5 Mathematical analysis for determination of the mutual lateral

diffusion coefficient
Fluorescence quenching in 2D was studied extensively[76] inspired by
Smoluchowskis fundamental work.[77] The complex expression based on integrals of
Bessel functions can be approximated[76b, 78] by employing the same functional form
as the exact decay law for 3D fluorescence quenching (see equation 1). The resulting
equation holds true especially for systems with fluorescence lifetimes in the range of
100 ns, which is an almost ideal match for Fluorazophore-L.
[
I(t) = I 0 exp ( k0 t + 2.31DL N a ) [Q2D ]t + 7.61 DL RN a [Q2D ] t ] (1)
variables are defined as follows: [Q2D] is the two-dimensional concentration

The
of the quencher (antioxidant) on the surface of the liposome with an assumed
arrangement of the polar headgroups towards the solvent, ranging generally from 0.5
x 1016 to 5 x 1016 molecules/m2 (corresponding to 0.5-3 mol%). The area per
molecule POPC and DPPC was taken to be 70 2 or 63 2, respectively.[12b, 66, 79] R is
the intermolecular distance at which fluorescence quenching occurs, taken to be 8
for -tocopherol.[31a, 70] I0 and I(t) are the fluorescence intensities at the time 0 or time
t, respectively. k0 is the inverse lifetime in the absence of additives (1/0), the
unquenched decay rate.
Chapter 3
Investigation of Antioxidant and Probe Structure
as well as their Mobility and Position
Chapter 3: Antioxidant Activity Measured by Fluorescence 35
Antioxidant and Probe Structure and Function
3 Antioxidant Activity Measured by Fluorescence:

Investigation of Antioxidant and Probe Structure as
well as their Mobility and Position
3.1 Analysis of a synthetic analogue of -tocopherol
Corresponds to Appendix II.
Tetrahydro-1,8-naphthyridinol Analogues of -Tocopherol as Antioxidants in

Lipid Membranes and Low-Density Lipoproteins
T. Nam, C. L. Rector, H. Kim, A. F.-P. Sonnen, R. Meyer, W. M. Nau, J. Atkinson, J.

Rintoul, D. A. Pratt, N. A. Porter, J. Am. Chem. Soc. 2007, 129, 10211.
That the antioxidant activity of -tocopherol is intimately related to its easily

abstracted H-atom, due to its low H-bond dissociation enthalpy (BDE), is a known
fact. Hence, the development of -tocopherol derivatives with even lower BDEs, and
consequently better antioxidant activity, has become an interesting field of study.
Several compounds closely related to -tocopherol on a structural level, the
naphtyridinols, were synthesized by the workgroup of Prof. Dr. Porter. These have
shown an up 25 times larger inhibition rate constant for chain-propagating radicals
than -tocopherol.[80] Of these compounds, the derivative with a lipophilic phytyl
sidechain similar to -tocopherol was dubbed N-TOH (naphtyridinol based
tocopherol, see Scheme 3.1) bore the closest resemblance to actual tocopherols. The
joint effort of all the authors of the article investigated the antioxidant activity and
function of N-TOH as an inhibitor of low-density lipoprotein degradation,[13c, 81] its
ability to bind to human tocopherol transfer protein (hTTP),[82] and its lateral
diffusion in POPC liposomes. The latter was the contribution of our workgroup and
shall be discussed below.
Scheme 3.1: Naphtyridinol based tocopherol (N-TOH)
HO
N N
N-TOH
-Tocopherol is known to not be a particularly potent antioxidant in LDL particles.

In fact, without co-antioxidants such as vitamin C, for example, it even has been
shown to have adverse effects and induce radical formation, albeit at a slow rate.[81b]
Supplementation of freshly prepared plasma with N-TOH showed a dramatic
difference in the oxidation profile of cholesteryl linoleate, efficiently precluding
oxidation and sparing endogenous -tocopherol, which decreases instantly in the non-
supplemented case. When N-TOH is completely used up, at around 5h for a
concentration of 75 M, the rate of cholesteryl linoleate oxidation increases
significantly, as does the degradation of -tocopherol.
The tocopherol transport protein plays an important role in supplying -tocopherol

to fluids and tissues in the human body.[23] In order for a synthetic compound to be a
viable substitute for -tocopherol it needs to be able to bind to hTTP, which was
tested by using an NBD-labeled -tocopherol compound. N-TOH is an even better
competitor for the bound fluorophore than -tocopherol. Under the experimental
conditions it took 2 M of N-TOH to reduce the fluorescence of the TTP/NBD-Toc
to 50% of its starting value, while 3.2 M of -tocopherol were needed to archive the
same effect. Furthermore, an X-ray crystallographic structure examination revealed
an additional H-bond between the N-CH3 moiety and the Ser-136 of hTTP, more than
offsetting the loss of the van der Waals interactions accrued due to the replacement of
the C(8)-methyl group by a nitrogen lone pair, leading to the preferential binding of
N-TOH over -tocopherol.
Our contribution to the article is based on the measurement principle for

determining the mutual lateral diffusion coefficient, which is described in Section
1.6.2. In case of N-TOH, the fluorescence-quenching rate constant in benzene was
determined, which allows another approach to determine the hydrogen donation
propensity, based on the BDE, of any antioxidant. The value determined for N-TOH
(kq = (5.4 0.5) 109 M1s1) is very similar to the one for -tocopherol (kq = (5.3
0.3) 109 M1s1); both are close to the diffusion-controlled limit. To assess the
mobility in and the accessibility of N-TOH for radicals inside lipid assemblies, the
previously described methodology, i.e., ethanol-injection POPC liposome generation,
single-photon counting measurements at varying antioxidant concentrations and
global fitting of the obtained data, was performed. The results reinforce the theory
that substitutions in the chromanol headgroup can indeed lead to lipid-soluble

antioxidants that are even more potent than the naturally occurring forms. While the
DL values are slightly higher for N-TOH than for -tocopherol (5.4 107 cm1 s1 vs.
3.73 107 cm1 s1 at 40 C)[21], the intrinsic fluorescence of N-TOH hindered the
method to some degree, resulting in more complex fitting and slightly elevated
experimental errors (see Fig. 3.1). The higher average mutual lateral diffusion
coefficient, with a similar activation energy of 43 6 kJ/mol (-tocopherol 47 5
kJ/mol), is indicative of a higher overall antioxidant potency, perhaps due to a better
positioning of N-TOH in the liposomal membrane.
Figure 3.1: N-tocopherol lifetime trace with quencher, note the bi-exponential
lifetime
In conclusion N-TOH is a highly functional synthetic lipophilic antioxidant, as

indicated by its good oxidation protection in LDL particles, its excellent mutual
lateral diffusion coefficient, and its ability to bind, and thus be transported by, hTTP.
These experiments also highlight the importance of fundamental research into lipid
dynamics, such that synthetic compounds could be compared to naturally occurring
ones.
3.2 Effects of substitution on DBO fluorescence

Corresponds to Appendix III.
Effect of bridgehead substitution on the fluorescence quenching of

2,3-diazabicyclo[2.2.2]-oct-2-enes by solvents and antioxidants
R. Meyer, X. Zhang, W. M. Nau, Photochem. Photobiol. Sci. 2009, 8, 1694.
The unique photophysical properties of DBO and its derivative Fluorazophore-L

have already been discussed (see Section 1.5.1). Due to these properties, derivatives
of DBO are valuable compounds for use as probes. Designing DBO-probe/quencher
systems to tailor them to the specific needs of a certain photophysical demand is
achieved by substitutions at the bridgehead position of the molecule.[59, 64, 83] While
certain substitutions lead to undesirable results, i.e. the shortening of the fluorescence
lifetime for -delocalizing subunits,[83a, 83c]
others, for example with inductively
active alkyl or halogen substituents, can have beneficial effects.[64, 83c, 84]
We have
now compared the parent 2,3-diazabicyclo[2.2.2]-oct-2-ene, or DBO-P to 5-methyl-1-
isopropyl-2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO-S), which exhibits a lower
reactivity and a higher selectivity, and 1,4-dichloro-2,3-diazabicyclo[2.2.2]-oct-2-ene
(DBO-R) which displays an increased reactivity, an even longer lifetime in water and
a decreased susceptibility to oxygen quenching. These three fluorescent probes (see
Scheme 3.2), which were synthesized and investigated by Dr. X. Zhang in a
preliminary fashion during his Ph. D. work,[85] were tested with various quenchers,
and the changes in their photophysical properties were carefully monitored.
Scheme 3.2: Fluorazophores
Cl
N N N
N N N
Cl
DBO-P DBO-S DBO-R
The photophysical properties of fluorescent probes depend strongly on the solvent

environment. It is therefore of the utmost importance to analyze these solvent-
dependent effects to assess their day-to-day experimental worth. A sufficiently long
lifetime in a certain solvent is the prerequisite for dynamic quenching experiments,
for example. Hydrogen-containing solvents interact with bicyclic azoalkanes by
aborted hydrogen atom donation, verified by pronounced deuterium isotope effects,
i.e. the longer lifetimes in deuterated solvents (see Table 3.1).
Table 3.1: Lifetimes of the azoalkanes in various solvents
Nonprotic /ns Protic /ns
solvent DBO-P DBO-S DBO-R solvent DBO-P DBO-S DBO-R
Gas phase 915 1150 --- D2 O 730 810 750
Freon-113 550 --- 473 D2O (air) 505 590 d 705
CD3CN 825 1060 610 H2 O 420 340 485
CH3CN 690 970 545 H2O (air) 325 290 465
C6H6 455 770 47 CH3OH 22 68 22
n-hexane 335 770 30
CH2Cl2 185 450 82
CCl4 72 12 d 403
CDCl3 110 275 76
CHCl3 13 65 12
The four main driving forces that determine the quenching are steric arguments,
the shift in absorption maxima, the nucleophilicity and the presence of charge-
transfer induced quenching.
The long lifetime of DBO-P[86] in aerated solvents arises due to its small singlet-
triplet gap (20-23 kcal mol1) which does not allow quenching by energy transfer, and
an oxidation potential that is too high to allow electron transfer processes (EP = 1.45
V vs. SCE).[87] This leaves only oxygen-assisted intersystem crossing (ISC) as the
main interaction channel. The low bimolecular oxygen quenching rate constant for
DBO-R (320 106 M1 s1, 6-8 times smaller than those of DBO-P and DBO-S)
confirms the notion that charge-transfer interactions can contribute significantly to
the quenching by oxygen.
All investigated azoalkanes display significant quenching by hydrogencarbonate

HCO3, with rate constants of up to 106 M1 s1. The O-H bond dissociation energy
lies at 95 kcal mol1, lower than water, and thus in the range where hydrogen-atom
abstraction as the quenching mechanism is feasible from a thermodynamic point of
view. The reactivity pattern of the three investigated compounds suggests a mixture
of factors determining the quenching rate constants, namely steric effects and
nucleophilicity.
The reactivity of the antioxidants towards azoalkanes follows a distinct trend: -

tocopherol > uric acid > ascorbic acid > reduced glutathione >> -D-glucose,
according to their O-H, S-H or C-H BDEs. No difference is observed here for the
three azoalkanes. This implies that hydrogen atom abstraction is the common
quenching mechanism (see Section 1.5.1). The azoalkanes are good examples for the
selectivity-reactivity principle, in which a more reactive probe (higher kq) shows a
lower selectivity (less variation in overall rates between different quenchers), and
vice versa. This phenomenon, in addition to its relative insensitivity towards oxygen
quenching and long lifetime in water, renders DBO-R particularly useful in the
elucidation of quenching by less active antioxidants, e.g., glutathione.
Lactone-based antioxidants have proven their usefulness in the evaluation of the

philicity of reactive species. Two 3-aryl-3H-benzofuran-2-one compounds, one
substituted with an electron withdrawing (p-cyano) and one with an electron-donating
(p-methoxy) group, have been employed to assess the philicity of the fluorazophores.
Singlet-excited DBO-P and DBO-S must be qualified as nucleophilic species,
whereas DBO-R is philoneutral or even mildly electrophilic.
The family of fluorazophores shows a lot of potential as probes for fluorescence

detection or measurement systems, due to their dramatic changes in photophysical
properties accrued due to bridgehead substitution. DBO-R is an attractive probe in
applications where a high reactivity, i.e. large overall quenching effect, is desirable,
or if oxygen quenching would pose an insurmountable challenge. Its lower
nucleophilicity in the excited state, the cause for the exceedingly long lifetimes in
water, was also independently ascertained by quenching experiments with substituted
lactones.
3.3 Phase dependence of antioxidant diffusion

Corresponds to Appendix IV.
Phase-Dependent Lateral Diffusion of -Tocopherol in DPPC Liposomes

Monitored by Fluorescence Quenching
R. Meyer, A. F.-P. Sonnen, W. M. Nau, Langmuir 2010, 26, 14723.
The elucidation of the inner workings of low molecular weight chain-breaking

antioxidants (see Section 1.4) via the DBO-based fluorescence detection method (see
Section 1.5.1.1) is still in its infancy. An important extension of the previous
investigations[21, 31a]
was the transition from a monounsaturated to a fully saturated
lipid, i.e., from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). This change allows the
investigation of phase-transition phenomena and their influence on vitamin E
diffusion, because DPPC has a readily accessible phase-transition temperature of 41
C (vs. 2 C for POPC).[88] This leads to DPPC adopting three main phases. Above
41 C the lipid exists in the liquid-crystalline phase, in which diffusion is assumed to
be very similar to that in POPC membranes. Below 32.5 C,[89] the lipid is in its rigid
solid-gel phase, making diffusion an extremely slow process. Lastly, a ripple phase is
found between 32.5 C (the so-called pre-transition temperature) and the main phase
transition at 41 C. Conceptually, part of the liposome is already melted, while the
rest is still found in the solid-gel state. The melted parts are believed to span rings
around the circumference of the liposome, in order to minimize steric stress and to
keep the overall geometry as constant as possible (see Fig. 3.2).
Figure 3.2: Schematic representation of the phases in DPPC liposomes with respect
to dependence on temperature, where the patterned grey and blue regions signify rigid
and more fluid regions with negligible or sizable diffusion, respectively.
As our measurement system does not yield structural data, the interpretation is
done in a way that explains the observations in the most elegant and plausible way
possible.
The determination of the fluorescence lifetimes of the DBO-probes is imperative,

not only as a key parameter for the global fitting (see Section 2.5), but also to check
whether the global fitting function, which is optimized for decays of approximately
100 ns, is applicable at all, as well as the information if the decay is mono- or
biexponential. The latter can accurately indicate in which phase the liposome is as
monoexponential decays are exclusively found in the liquid-crystalline phase,
whereas the preexponential factors can even afford the pre-transition temperature
rather accurately, as determined by DSC. It bears mentioning that the pre-transition,
as well as the main phase transition temperature, is depressed. This is due to the
addition of probe and quencher to the mixture (pre-transition at approx. 30 C, main
phase transition at 38 C, see inset of Fig. 3.3).
Three different ranges for the mutual lateral diffusion coefficients DL were
identified. In the solid-gel phase, below 30 C, the DL can be assumed to be smaller
than 109 cm2 s1, below the detection limit of our method. Above the main phase
transition temperature, in the liquid-crystalline phase, DL values of 107 cm2 s1 are
found, analogous to POPC, albeit slightly lower. The third, with DL values of 108
cm2 s1, is the ripple phase, in which only those antioxidants and probes located in the
already melted regions, the ripples, can diffuse with relative ease, while those still
located in the solid-gel like part of the liposome do not move at all. In order to
calculate the DL in the ripple phase, the original equation (see Section 2.5) had to be
expanded (see equation 2 and the corresponding simplified equation 3).
exp k t + 2.31D N [Q ]t + 7.61 D RN [Q ] t

1 [( 0,1 L1 a 2D L1 a 2D ) ] +
I(t) = I 0 (2)
exp k t + 2.31D N Q t + 7.61 D RN Q
2 [( 0, 2 L2 a [ 2D ] L2 a [ 2D ] t ) ]
exp k t + 2.31D N [Q ]t + 7.61 D RN [Q ] t

I(t) = I 0
1 [( 0,1 L1 a 2D L1 a 2D
)] (3)
+2 exp( k0, 2 t)

The recovered DL values were in good agreement with the previously published
literature values, both by us and by others (see the full article, Appendix IV). For
DPPC in the liquid-crystalline phase the values are close to the ones for POPC, and
the slopes are almost parallel (see Fig. 3.3). This leads to essentially identical
activation energies (49 5 kJ/mol for DPPC vs. 47 5 kJ/mol for POPC). In the
ripple phase, the activation energy is much higher, 175 50 kJ/mol.
Figure 3.3: Arrhenius plots of the mutual lateral diffusion coefficient of

Fluorazophore-L with -tocopherol in DPPC liposomes (, data points below the
phase transition temperature are shown with associated errors) and POPC liposomes
(,[21] data point at 15 C is cut out for clarity). Note the different behavior below
and above the phase transition temperature, for which two different linear regression
lines are drawn. The inset shows DSC curves for DPPC liposomes (lipid
concentration 0.7 mM) without additives (red), with 70 mM Fluorazophore-L (green),
and with 70 mM Fluorazophore-L and 23 mM -Toc (blue)
In conclusion, Fluorazophore-P is a very good probe for antioxidant diffusion in

liposomes. Even the relatively slow processes in the ripple phase can readily be
investigated, with only minor tweaks to the overall method. The lateral diffusion
coefficients recovered were reproducible (independent experiments over a time span
of 1 year), and also for different batches of lipid and over a wide temperature range.
Antioxidant & Probe Structure and Function
3.4 pH dependence of L-ascorbyl-6-palmitate antioxidant activity

The biological relevance of lipophilic antioxidants has been discussed previously
(see Section 1.4). They are also of interest for industrial applications, for instance in
cosmetics[90] or the food industry,[36b] or even as a counteragent to plastic bleaching.
The biologically most relevant class, the vitamin E constituents, have one major
downside from an industrial point of view, namely their cost. Therefore, ascorbyl-6-
palmitate (A6P) is often used in its stead, which is a fatty acid linked via an ester
bond to ascorbic acid (one of natures foremost water-soluble antioxidant). Its several
advantages include the lower price as well as the fact that it is not inherently
hazardous to humans. It is marketed as antioxidant food additive E304 and is widely
used in products such as sausages, mayonnaise, or chicken broth, but also as a cheap
facsimile for vitamin E in budget skin ointments or sunscreen.
As outlined in Section 1.6.2 the diffusion studies yielded only a very low diffusion
coefficient of A6P inside POPC liposomes, whereas the diffusion in solution is very
similar to tocopherols and tocotrienols. This does not necessarily mean that A6P does
not diffuse as well as the vitamin E constituents in liposomes, since the assay
procedure is based on a contact quenching of the antioxidant with the probe
Fluorophore-L. The aborted hydrogen abstraction mechanism, by which the n,*
excited state of DBO is quenched, has an interaction radius of approximately 8 , i.e.
the probe and quencher need to be in very close proximity. This means, by default,
that anything changing the penetration depth of the antioxidant also influences the
encounter probability of probe and quencher, and, thus, the extracted mutual lateral
diffusion coefficient.
Ascorbic acid, and A6P accordingly, has a pKa of 4.25. This means that at pH 7 a
high amount of the A6P molecules will be deprotonated and thus charged. This may
lead to a dislocation from the membrane to the surrounding water. If the A6P
molecule sticks out of the membrane, it will of course not be able to undergo
contact quenching with the Fluorazophore-L, and therefore only the uncharged
molecules will quench the fluorescence (see Figure 3.4). This leads to a lower
observed lateral diffusion coefficient, and even though it is not an indicator of actual
lateral diffusion, it is an indicator of how well the antioxidant is able to interact with
radicals.
pH 7
pH 3
Figure 3.4: Placement of the A6P molecule (red) at high and low pH. Probes,
Fluorazophore-L and Fluorazophore-LE, shown in blue.
In order to verify this hypothesis, quenching experiments at varying pH have been

performed. Our analysis system has previously been shown to yield reliable data in
determinations of two-dimensional diffusion, so we proceeded to use Fluorazophore-
L as the probe, this time with A6P as quencher in POPC liposomes. In order to prove
our working hypothesis that a lower pH of the medium will lead to better penetration
depth and more quenching, an experiment at pH 3 was performed. During the course
of these measurements it became obvious that our probe did not meet the demands
imposed on it by the low pH environment. In contrast to the mono-exponential decay
traces at neutral pH, a bi-exponential decay was observed. One of these lifetimes was
approximately the same length that is observed for pure DBO in water.
Experiments were performed in which Fluorazophore-L was incorporated into

POPC liposomes and then kept at pH 2 for a prolonged time. While biexponential
from the start, the longer component became more and more pronounced over time.
This longer component is very similar to the lifetime of DBO-OH in solution (see
Figure 3.5). The most probable explanation for this is the acid catalyzed hydrolysis of
the ether linking the C16 tail to the DBO headgroup. In order to remedy this situation,
a novel probe was designed and successfully synthesized in which the two subunits
are linked by an ether bridge, which we dubbed Fluorazophore-LE. No bi-
exponentiality was observed for the ether linked probe at pH 2, even in a time frame
many times that of our measurement time frame (see Figure 3.5).
100
DBO-OH
101
Normalized Counts
102 Fluorazophore-L
103
Fluorazophore-LE
104
0 200 400 600 800 1000
! in ns
Figure 3.5: Lifetime decay traces of various DBO derivatives at pH 2. DBO-OH
traces recorded in water. Fluorazophore-L traces recorded in POPC liposomes, with
progressing time (light color to darker color), directly after liposome creation, after 4
hours, 8 hours and after 20 hours at 50 C. Fluorazophore-LE traces were recorded
after 20 hours at 50 C.
Preliminary experiments with the new probe suggest that the observable mutual
lateral diffusion coefficients are one order of magnitude greater at lower pH than at
pH 7, very similar to those recovered for the vitamin E constituents. This would mean
that A6P is an efficient lipid antioxidant only in acidic environments, and should only
be used as such if the pH value is low enough, such that A6P predominantly adopts
its neutral form. At neutral pH vitamin E should be used as a lipid-soluble
antioxidant. We could also successfully increased our toolbox of available probes by
the addition of an acid-stable lipid-soluble derivative, Fluorazophore-LE, which has
been synthesized by Dipl.-Chem. T. Schwarzlose.
3.5 Conclusions
During the course of this Ph.D. work it could be shown that DBO based fluorescent
probes are especially suited to investigate antioxidant behavior. Dyes from the
fluorazophore family allow both the determination of the hydrogen atom donor
propensity of an antioxidant, as well as the mutual lateral diffusion coefficient of a
lipid soluble antioxidant in membrane systems. The methodology is sensitive enough
to allow even the measurement of diffusion processes in the ripple phases of
membranes, with mutual lateral diffusion coefficients as low as 0.2 108 cm2 s1.
The diffusion of antioxidants in the ripple phase has been studied with fluorescence
for the first time. The high degree of modularity of the dye, afforded by the
substitution at the bridgehead position, is beneficial for other purposes as well, for
example in acidic media, made possible by changing the ester to an ether linkage.
Additionally, it could be demonstrated that the lifetime of the DBO dyes can yield
additional information about the position or environment of the probe, for example in
a lipid system. It is now possible to elucidate synthetic antioxidants as well, because
they can be compared to the naturally occurring ones. This yields, for the first time,
an accurate assessment method to gauge the potency of a synthetic antioxidant.
3.6 Perspectives
There are several promising routes to take in order to advance the projects conducted
in this Ph.D. work. One of the first steps would be to determine the individual lateral
diffusion coefficient of Fluorazophore-L. This would allow the determination of
lateral diffusion coefficients of antioxidants, without the need to factor in the
diffusion of the probe as well. The most promising approach for this problem would
be to take a fluorescent probe with a known lateral diffusion coefficient (NBD-
derivatives, for example) and to cross-correlate this with our mutual lateral diffusion
coefficients. Then, our methodology can be expanded to different lipids
(phosphoethanolamines or sphingomyelins) or to liposomes composed of multiple
lipids (POPC and DPPC, for example), in order to investigate situations similar to the
lipid raft formation observed in biological membranes. Also, the addition of
biologically relevant membrane additives, for example membrane-bound proteins,
channels, or protein anchors, could yield valuable insights into in vivo membrane
function. Other relevant antioxidants, like curcumin, could also be investigated with
our method. A long-term goal would be to determine another important process in
membranes, the flip-flop, or transversal diffusion, where a molecule changes
between the inner and outer leaflet of the membrane. To measure this very slow
process, substantial alterations to our measurement method will have to be made. One
idea would be to destroy the fluorescent probe in the outer leaflet, and measure the
destruction of flip-flopping probes over time. The time-range necessary for these
measurements is quite large, as transversal diffusion occurs over multiple hours or
even days. This might be remedied by employing bolaamphiphiles, be they simple
molecules or membrane spanning peptides, which facilitate transversal diffusion.
Chapter 4
Industry Project
Chapter 4: Industry Project 57
4 Industry Project
I have also been involved in an industry project during my Ph.D. regimen. I
remain under a non-disclosure agreement, so I will only give general information
regarding the goals of the study and the experiments performed in order to achieve
them.
The company that commissioned the survey had developed a food additive, based
on various ingredients found in fruit and vegetables that should be able to increase the
antioxidant potential in humans. The objective of the test was to determine how a pill
containing these ingredients would alter the antioxidant potential and population in
the body, or more specifically, in bodily fluids. In order to accomplish this, blood
serum and urine samples were analyzed for their antioxidant content or antioxidant
potential.
4.1 Samples
The samples were collected by qualified medical personnel and shipped to our
laboratory on dry ice. These samples were anonymous, containing no information
about the donor or whether or not the person was part of the test or control group
(who were given placebos).
4.2 Parameter determination techniques

In order to assess all of the parameters that were desired by the client two major
experimental approaches had to be taken. In order to elucidate the blood serum levels
of vitamin E, vitamin C and uric acid, high performance liquid chromatography
(HPLC) was chosen as the best applicable procedure and was performed by A.
Mller. For the total antioxidant power of the blood serum, the reduced glutathione
(GSH) concentration therein and the isoprostane amount, as an indicator of oxidative
stress in urine, as well as the concomitant determination of creatinine, standardized
sample analysis-kits were purchased.
4.3 Vitamin E
For a description of vitamin E as an antioxidant see Section 1.4.1. The method
used for HPLC determination of vitamin E employed a Polaris Ether-RP-18-A-
250/4.6/5 column with an oven temperature of 25C and a flow of 1.0 ml/min at the
monitoring wavelength of 280 nm. The eluent was a 75% acetonitrile/25% methanol
mixture. The samples were prepared by diluting 100 l sample with 400 l ethanol in
order to precipitate the proteins. After centrifugation for 6 min, 10 l of the
supernatant were injected. If the sensitivity was to low, the injection amount was
increased to 20 l.
4.4 Vitamin C and Uric Acid

Uric acid is generated by oxidization of xanthine or hypoxanthine by various
enzymes. The peroxisomal enzyme urate oxidase converts uric acid to allantonin.
However, this enzyme is deactivated by a stop codon in one of the exons that inhibits
transcription in primates. Therefore, uric acid is present in much higher
concentrations in primates, a condition that can lead to illnesses such as gout (due to
the poor water solubility of uric acid it accumulates in the joints and can cause
inflammation). As the antioxidant with the lowest reduction potential,[32c] it is the one
consumed first in cases of intense oxidative stress, i.e., it is able to shield the other
antioxidants.[1, 91] For information on vitamin C, see Section 1.4.2.
For the parallel HPLC determination of uric acid and ascorbic acid the column
used was a Polaris Ether-RP-18-A 250/4.6/5, 20 mmol/L KH2PO4, 3.9 mmol/L
phosphoric acid and 5% acetonitrile eluent-mixture. At a 1.0 ml/min flowrate 10L
sample were injected and measured at 265 nm at 25C oven temperature. Sample
preparation consisted of mixing 200 L sample with a 200 L orthophosphoric acid
and perchloric acid mixture (6:3), after which 20 mmol/L of
ethylenediaminetetraacetate (EDTA, to prevent transition metal catalyzed
deactivation of the antioxidants) was added. This mixture was centrifuged for 6
minutes and 10 L of the supernatant was measured. If the signal was too low, a 20
L sample was injected.
4.5 Total antioxidant power[92]

The total antioxidant power assay kit employed is based on a simple principle. The
action of the antioxidants in the sample reduces Cu++ to Cu+. The reduced copper will
then form selectively a 2:1 complex with the chromogenic reagent. This complex is
stable and can be measured at 490 nm in a multiplate reader equipped for 96-well
plates. With this relatively simple approach it is possible to assess the total
antioxidant defense systems in a biological system (see. Section 1.3) and to report
data as millimolar uric acid equivalents (uric acid being used as the reference).[32c]
The kit used was the Oxford Biomedical Research Total Antioxidant Power Assay kit
TA 01.
4.6 Isoprostane[93] and creatinine[94]

Isoprostanes are a family of eicosanoids of non-enzymatic origin, produced by
random oxidation of tissue phospholipids by reactive oxygen species (see Scheme
4.1).
Scheme 4.1: Generation of isoprostane
H 2C O Fatty Acid
HC O Arachidonate R=(CH2)2N(CH3)2
H 2C O PO3 R+
-O2 or R
O
H 2C O Fatty Acid
!-Cleavage H
HC O Peroxy Acid
OH
H 2C O PO3 R+
4-hydroxy nonenal and other aldehydes
Rearrangement
COOH
H 2C O Fatty Acid O O
HC O Endoperoxide
H H
H 2C O PO3 R+ malondialdehyde
Reduction by 12(S)-HHTrE
cellular [12(S)-HHT]
OH
peroxidases OH
H 2C O Fatty Acid
Phospholipas A2
HC O Isoprostanes COOH
H 2C O PO3 R+
HO
OH
8-Isoprostane (8-epi PGF 2")

+
other cis- side-chain isomers
+
lyso-phospholipids
In contrast to the other factors determined in this study, isoprostane is not a

beneficial antioxidant but rather an indicator of oxidative stress. The higher the level
of isoprostane in the bodily fluids the more oxidative stress is put on the organism;
elevated levels are for example found in smokers. 8-isoprostane (8-epi-PGF2) has
also been shown to have physiological activity as a potent pulmonary and renal
vasoconstrictor. The kit used is an enzyme-linked immunosorbent assay (ELISA)
from IBL, CM 516351. It is based on the competitive binding of 8-isoprostane and a
tracer (8-isoprostane covalently linked to acetylcholinesterase) to a rabbit anti-8-
ELISA!
isoprostane antibody. The amount of tracer able to bind to the antigen binding sites
on the antibody is inversely proportional to the concentration of 8-isoprostane in the
sample. The 96-well plate is coated with mouse anti-rabbit antibodies that bind to the
Enzyme Linked Immunosorbent Assay!
Fc-portion of the rabbit antibodies (see Scheme 4.2).
Scheme 4.2: 8-Isoprostane ELISA
Mouse monoclonal antibody!
Free 8-Isoprostane!
Mouse Monoclonal Antibody!
Acetylcholinesterase linked !
to 8-Isoprostane (Tracer)!
After a wash step Ellmans Reagent is added,[95] consisting of acetylthiocholine

and 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, see Scheme 4.3). Hydrolysis of
acetylthiocholine by acetylcholinesterase produces thiocholine, which reacts non-
enzymatically with DTNB to yield 5-thio-2-nitrobenzoic acid (TNB) that displays a
strong absorbance at 412 nm.
Scheme 4.3: Reaction scheme for the isoprostane ELISA
O
N Acetylthiocholine
S
Acetylcholinesterase
O
N Thiocholine
O S
NO2 S S NO2 5,5'-Dithio-bis-

(2-nitrobenzoic acid)
OOC COO
NO2 S N S NO2
S
OOC COO
5-Thio-2-nitrobenzoic acid
It is not possible to directly use the data recovered by the 8-isoprostane assay. This
is due to the widely varying behavior of the test subjects. For example, a person who
drinks a lot of fluids will produce more urine, so the concentration of 8-isoprostane
will be lower. To account for this, a concomitant creatinine assay has to be
performed. The usual unit used to report urinary 8-isoprostane levels is ng/mmol
creatinine.
Creatine, produced in the kidney, liver, and pancreas is used in brain and muscle
cells, where it is phosphorylated and used as an energy source.[96] A small portion is,
however, converted irrevocably to creatinine, in relation to the muscle mass of the

individual. Daily excretion is relatively constant, so creatinine is a valuable
compound as an index of standardization. The assay used, the Creatinine CR01 assay
by Oxford Biomedical Products, is based on a simple colorimetric principle. It is
based on the so-called Jaffe reaction,[97] which remains, 125 years after its discovery,
of experimental relevance.[98] Creatinine reacts with picric acid (see Scheme 4.4)
under alkaline conditions to produce an orange compound that can be quantified by
absorption spectroscopy at 500 nm. Even though this reaction occurs non-specifically
with other components of biological samples, the specific color given by creatinine
degrades rapidly under acidic conditions.[99] Measuring the absorption before and
after addition of acetic acid,[100] it is possible to accurately determine the urinary
creatinine levels quickly and reliably.
Scheme 4.4: Structures of a) creatinine and b) picric acid
a)
N N
NH NH2
N N
O O
H
OH
O2N NO2
b)
NO2
4.7 GSH[101]
GSH is a tripeptide (-glutamyl-cysteinylglycine) and the major free thiol in most
living cells. It is involved in a variety of biologically important processes and the
major antioxidant in animal tissues.[102] Most of the cellular glutathione is in its
reduced form (90-95% of the total glutathione). Oxidation leads to the formation of
glutathione disulfide, GSSG. In addition to being an important indicator of overall
cell health, elevated levels of GSH may indicate pathological changes in biological
samples.[103] The Glutathione Assay Kit CS0260 by Sigma employed here uses the
conversion of DTNB by GSH to a yellow product, TNB, which can be quantified by
absorption measurements at 412 nm with a kinetic read out. GSH is hereby
regenerated from GSSG by glutathione reductase and the reduced form of
nicotinamide adenine dinucleotide phosphate (NADPH) (see Scheme 4.5)
O O NO2 O O O O
C OOC C C
H C H H C H H C H
N H N H H N
S
O C H O C H H2 C O
2 S 2
2 H C C SH
S
H C C S S C C H 2
N H N H H N
OOC
O C O C C O
NO2
CH2 CH2 CH2
CH2 OOC CH2 CH2
NO2
H3N C H NH3 C H H C NH3
C C C
O O 5,5'-Dithio-bis- O O O O
5-Thio-2-nitrobenzoic acid
(2-nitrobenzoic acid)
Scheme 4.5: Colorimetric GSH assay principle
Because the conversion rate is proportional to the GSH content up to very high
levels, it is a reliable way to elucidate the total glutathione content of a sample.
Because of the low content of GSH in blood serum, it became necessary to measure
all samples individually on a Cary 4000 UV spectrophotometer; the multiplate reader
did not offer sufficient resolution to guarantee accurate results.
Glutathione reductase
GSSG + NADPH + H+ 2 GSH + NADP+
References 65
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Chapter 5
Appendix
Chapter 5: Appendix I 73
Global Fitting Procedure
Appendix I: How to perform a global fit to determine DL
The lifetimes are recorded in the same time frame, 40 min, and with the exact
same slits. Each fluorescence decay trace is exported from the Edinburgh Instruments
software as a .txt file. The global fit is based on data at the same temperature, but
with different quencher (i.e. antioxidant) concentrations. It is helpful to make new
folders for each temperature and copy the corresponding traces at the varying
temperatures (hereafter referred to as Sn, where n is the amount of quencher added to
the mixture in the experiment, Ref is the reference measurement containing neither
probe nor quencher) into them, labeling them according to temperature.
Now it is time to create the Mastertable for each temperature, containing all the
information needed to proceed with the global fitting. The function New Data is
used to bring up a blank data file. A standard data file in ProFit contains 10 columns,
which is insufficient. After marking the rightmost column, Calc and Insert
Rows/Columns will bring up a window. Checking Insert at the right of selection,
and inserting 10 additional columns will be sufficient. The columns are named as
shown in Table 5.1. Additional data measured, i.e. S4, S6 etc., should be included as
well (for example, a setup with 4 concentrations should consist of 4 Sn values, 4 Sn
Ref columns and 4 Error Sn columns).
Table 5.1: Example of a Mastertable
Time/ns Time/s Sn1 Sn2 Ref Sn1Ref Sn2Ref Error Sn1 Error Sn2
... ... ... ... ... ... ... ... ...
... ... ... ... ... ... ... ... ...
The .txt files are then imported into ProFit, but care has to be taken to use the
correct delimiters. The imported files tend to contain file information that is not
necessary, so the real data trace is cut out and pasted into the corresponding column
in the Mastertable, see Table 5.2.
Table 5.2: Parts of the imported data traces to be discarded
Labels 060221DPPC_a-Toc_S0_48
Type Time Scan
Comment
start(nm) 0.00
stop(nm) 999.511.720 unnecessary
step(nm) 0.488281
fixed/offset(nm)
Xaxis Time
Yaxis Counts
0.00 1,00E+04
0.488281 1,00E+04
0.976563 1,00E+04
1.464.844 7,00E+03
1.953.125 1,00E+04
The Time/ns column need only be pasted once, as the time information is the
same for all traces. After all the experimental information has been compiled into the
Mastertable, convert the Time/ns column to the Time/s column. The option Data
Transform in the Calc tab is selected, minding the right in and out columns,
and with Simple Arithmetics the Time/ns column is multiplied by 109. Next the
reference values are subtracted from the decay traces. Again, Data Transform from
the Calc tab has to be selected, this time with Column Artihmetics, substracting
Ref from all the Sn columns, yielding the SnRef columns.
In order to calculate the individual error for each data point, the square root of the
unmodified traces, i.e. not subtracting the reference, is calculated. To each decay
traces Sn, +1 is added, again via Calc, Data Transform and Simple
Arithmetics, taking the same Sn column as the input and output columns, i.e.
overwriting the columns, then in the same window Various Functions and sqrt(x)
are used to calculate the error values Error Sn. The Mastertable is now complete
and should be saved.
The decay traces still contain information that is detrimental to the fitting
procedure, i.e. elastic scattered light and instrumental noise. That is why the first part
of the traces is discarded. The lifetime of the probe is sufficiently long to ensure
enough data stability, even if the first 100 ns are omitted from the global fitting. As a
general rule, all datapoints prior to the maximum have to be cut via Calc and
Delete Rows and Columns, and some more might be cut. As a rule of thumb,
anywhere between 70 ns to 100 ns will give a good fit.
A new file, the so-called Fitting-file, will now be created. This contains 5 columns:
N (which is just a running number), T/s, Counts, 2D-Conc. and Error (see Table 5.3).
Table 5.3: Sample Fitting-File
N Time/s Counts 2D-Conc Error
... ... ... ... ...
... ... ... ... ...

The data from the Mastertable are now pasted underneath each other for the
columns T/s (which is always the same), Counts (the SnRef columns) and Error.
The two-dimensional quencher concentration (2D-Conc) is calculated by determining
the number of quencher molecules per m2 of lipid. This is achieved by calculating the
amount of molecules in the experimental setup and multiplying this value by the area
per molecule (70 for POPC, 63 for DPPC and 34 for Fluorazophore-L). These
values are then pasted into the file corresponding to their decay traces (Sx). For
example, S0, the sample containing no quencher, will have a 2D-Conc of zero.
Now the part of the 2D-Conc-column referring to the S0 sample is highlighted,
Calc Data Transform is used and the tab Formula with the value 0 is used. In
order for this to be restricted to just the selected cells, the box Use selected rows
only has to be checked. This process is repeated for the other concentrations.
Finally, the row index is multiplied by 1 to give the N column, using Simple
Arithmetics from the Data Transform option. The file is now prepared for the
global fitting procedure and should be saved. Figure 5.1 shows an example how the
data should look like in the Preview window.
Figure 5.1: Example graph for global fitting
Fitting
Load the function, click To menu in the function window and switch to the
Parameters window (see below for an example of a function). Make sure the two-
dimensional quencher concentration in the function and in your Fitting file match. Put
in the initial values as follows: all cts=10000, all base=0, const1 and const2=0,
conc=0 (the last three are already integrated into the function), t0= the fluorescence
lifetime of the S0 trace as determined by the Edinburgh Instruments software, R=8
1010 (distance for contact quenching in ) and D=0. Now the following parameters
are deactivated by either left clicking on them or unchecking the box used for
fitting: t0, const1, const2, conc and R. The only parameters varied in the fitting are
now the counts of the traces, the baselines and our goal parameter, the mutual lateral
diffusion coefficient D, for which the lower limit should be set to 0 in the parameters
window. It is important to realize that the deactivated parameters, namely t0 and R,

still have to be correctly given, as a wrong value will cause the results to be erroneous
or the function to not fit at all. Open the Preview window and select N as the x-
and Counts as the y-column. Open Calc and Fit to go to the fitting window,
select Robust as the algorithm, fit. After the fitting procedure is completed, choose
Func Parameter Sets Fitted Parameters to import the fitted variables into the
parameter window as new initial parameters. Open the fitting window again, select
Levenberg-Marquardt as the algorithm and change for the Output data the error
type to Individual and the error column to Error. After the fitting is completed the
DL value is displayed in the Results window, together with a standard deviation. To
assess the overall goodness of the global fit, divide the chi2 value given in the results
window by the number of data points (highest number in the N column) used to
obtain the reduced chi2 value.
Sample function for DPPC with four different quencher concentrations (0,
1.714E+16, 3.428E+16 and 5.142E+16):
function User_Function (cts0;t0;baseline0;const1;const2;conc;base1;cts1;base2;cts2;base3;cts3;R;D);
begin
a[6]:=data[x,4];
a[4]:=7.61;
a[5]:=2.31;
if data[x,4]=0.00000 then
y:=a[3]+(a[1]*(exp(-(data[x,2])/a[2])))
else if data[x,4]=1.714E+16 then
y:=a[7]+(a[8]*(exp((-(data[x,2])/a[2])-(a[4]*a[6]*a[13]*sqrt(abs(a[14]*abs(data[x,2]))))-(a[5]*a[6]*a[14]*(data[x,2])))))
y:=a[9]+(a[10]*(exp((-(data[x,2])/a[2])-(a[4]*a[6]*a[13]*sqrt(abs(a[14]*abs(data[x,2]))))-(a[5]*a[6]*a[14]*(data[x,2])))))
y:=a[11]+(a[12]*(exp((-(data[x,2])/a[2])-(a[4]*a[6]*a[13]*sqrt(abs(a[14]*abs(data[x,2]))))-(a[5]*a[6]*a[14]*(data[x,2])))));
end;
Chapter 5: Appendix II, III and IV 78
Articles
Please find the articles mentionend in the previous chapters in their respective
journals:
T. Nam, C. L. Rector, H. Kim, A. F.-P. Sonnen, R. Meyer, W. M. Nau, J.

Atkinson, J. Rintoul, D. A. Pratt, N. A. Porter, Tetrahydro-1,8-naphthyridinol
Analogues of -Tocopherol as Antioxidants in Lipid Membranes and Low-Density
Lipoproteins, J. Am. Chem. Soc. 2007, 129, 10211-10219
R. Meyer, X. Zhang, W. M. Nau, Effect of bridgehead substitution on the

fluorescence quenching of 2,3-diazabicyclo[2.2.2]-oct-2-enes by solvents and
antioxidants, Photochem. Photobiol. Sci. 2009, 8, 1694-1700
R. Meyer, A. F.-P. Sonnen, W. M. Nau, Phase-Dependent Lateral Diffusion of -

Tocopherol in DPPC Liposomes Monitored by Fluorescence Quenching, Langmuir
2010, 26, 14723-14729
Chapter 5: Appendix V 127
Lebenslauf
AppendixV:Lebenslauf
Name: Roland Meyer
Geburtsdatum: 26.02.1978, Bremen
Schulische Ausbildung
1984 1988 Grundschule Philipp-Reis-Str.
1988 1994 Sekundarbereich I, Schulzentrum Ronzelenstr.
1994 1997 Sekundarbereich II, Gymnasium Horn
Abitur, Note: 2.1
Zivildienst
1997 1998 Hausnotrufzentrale der Arbeiterwohlfahrt, Bremen-Vahr
Studium
10/1998 07/1999 Studium der Chemie an der Universitt Bremen
10/1999 07/2002 Studium der Biologie mit Hauptfach Mikrobiologie an der

Universitt Bremen
Vordiplom, Note: 2.0
10/2002 04/2005 Studium im Masterstudiengang Biochemistry and

Molecular Biology an der Universitt Bremen
Master Thesis Structural analysis of CD22 and other

proteins of the SIGLEC family
Erworbener akademischer Grad: Master of Science
Note: 1.8
Lebenslauf
Mehrmonatige Forschungsaufenthalte whrend des Masterstudienganges
10/2003 12/2003 Max-Planck-Institut fr Marine Mikrobiologie Bremen,

Thema Effects of solvent pressure on Azoarcus strain
EbN1
01/2004 03/2004 Geesthacht, GALAB Laboratories GmbH, Thema

Determination of a suitable marker enzyme and
maximum plate coverage with a lectin
04/2004 07/2004 Jacobs University Bremen, Thema FRET distance

measurements of short, structureless polypeptides
08/2004 09/2004 Theoretische Ausarbeitung, sog. Project Proposal, eine

Einfhrung in das Schreiben von Antrgen, Thema DBO as
a means to analyze short -structural elements, especially -
hairpins
Promotion im Fachbereich Chemie an der Jacobs University Bremen
09/2005 Heute Doktorarbeit bei Prof. Dr. Werner M. Nau mit dem Thema
Investigation of Antioxidant and Probe Structure as well as
their Mobility and Position, Jacobs University Bremen
EDV Fhigkeiten
Word, Excel, Power Point
ProFit (Wissenschaftliches Kalkulationsprogramm)
EndNote
SciFinder
ChemDraw
Verschiedene online Datenbanken, z.B. RCSB Protein

Databank oder Expasy
DeepView/Swiss-Prot PDB viewer

Lebenslauf
Sprachen
Muttersprache: Deutsch
Fremdsprache: Sehr gutes Englisch in Wort und Schrift, Lehrsprache

whrend Masterstudium sowie Promotion: Englisch
Konferenzen und Workshops
NRP Final Symposium: Supramolecular Functional Materials, 16.06. - 18.06.2005, Murten

(CH)
ChemieContact Innovation sucht Partner des VCI (Verband der Chemischen Industrie
e.V.), 10.10.2006, Hamburg (D)
4th European short course on "Principles and Applications of Time-Resolved Fluorescence

Spectroscopy, 30.10. - 03.11.2006, Berlin (D)
21. Lecture Conference der Fachgruppe Photochemie, 06.10. - 08.10.2008, Bielefeld (D)
Bremen Molecular and Marine Biology Retreat, 26.01 27.01.2007, Etelsen (D)
NanoFun Center and Nanomol Graduate Retreat, 12.01 14.01.2011 Flammbacher Mhle,
Clausthal-Zellerfeld (D)
4. Treffen der Norddeutschen Biophysiker, 21.01.2011, Forschungzentrum Borstel (D)
Publizierte Artikel
T. Nam, C. L. Rector, H. Kim, A. F. P. Sonnen, R. Meyer, W. M. Nau, J. Atkinson, J.

Rintoul, D. A. Pratt, N. A. Porter
J. Am. Chem. Soc., 2007, 129 (33), 1021110219
Tetrahydro-1,8-naphthyridinol Analogues of -Tocopherol as Antioxidants in Lipid

Membranes and Low-Density Lipoproteins
R. Meyer, X. Zhang, W. M. Nau
Photochem. Photobiol. Sci., 2009, 8, 1694 - 1700
Effect of bridgehead substitution on the fluorescence quenching of 2,3-

diazabicyclo[2.2.2]oct-2-enes by solvents and antioxidants
Lebenslauf
R. Meyer, A. F.-P. Sonnen, W. M. Nau
Langmuir, 2010; 26, 14723 - 14729
Phase-Dependent Lateral Diffusion of -Tocopherol in DPPC Liposomes Monitored by

Fluorescence Quenching

Antioxidant Activity Measured by Fluorescence - 1col

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Antioxidant Activity Measured by Fluorescence:

Investigation of Antioxidant and Probe Structure as well as

A thesis submitted in partial fulfillment

Approved, Thesis Committee

Date of defense: 13.05.2011

School of Engineering and Science

Jacobs University Bremen, Germany

Optimisten sind Menschen,

List of Attended Conferences........................................................................................x

Abstract ..................................................................................................................... xiii

Chapter 2 Materials and Methods

Chapter 3 Antioxidant Activity Measured by Fluorescence: Investigation

Chapter 4 Industry Project

Finally, I am extremely grateful to my father, without whose constant support, both

R. Meyer, X. Zhang, W. M. Nau, Effect of bridgehead substitution on the

R. Meyer, A. F.-P. Sonnen, W. M. Nau, Phase-Dependent Lateral Diffusion of -

List of Attended Conferences

ChemieContact Innovation sucht Partner des VCI (Verband der Chemischen

4th European Short Course on "Principles and Applications of Time-Resolved

21. Lecture Conference der Fachgruppe Photochemie, 06.10. - 08.10.2008, Bielefeld

NanoFun Center and Nanomol Graduate Retreat, 12.01 14.01.2011 Flambacher

4. Treffen der Norddeutschen Biophysiker, 21.01.2011, Forschungzentrum Borstel

NADPH nicotinamide adenine dinucleotide phosphate (reduced)

1.1 Scope of the thesis

1.1.1 Summary and aims

membranes are considerable, due to the abundance of membrane additives in nature

1.2 Free radicals, and their importance in biological systems

Why do radicals receive so much attention? Their reactivity enables them to

As life evolved from basic unicellular bacteria to the conglomerates of millions of

1.3 Antioxidant defense systems: An overview

(1) Pro-oxidant restricting compounds, which minimize the availability of

(4) Low molecular mass compounds that scavenge reactive oxygen or

1.4 Low molecular weight chain-breaking antioxidants in and

Scheme 1.1: Structure of the vitamin E constituents

Vitamin E constituent R1 R2 R3 (isoprenoid tail)

!-Tocopherol CH3 CH3

The exact position that -tocopherol occupies in a membrane environment is not

Scheme 1.2: Location of -tocopherol in a membrane: a) near the interfacial region,

Scheme 1.3: Radical scavenging by antioxidants according to the floating radical

"Deactivation" of Regeneration of the !-tocopheroxyl

in collagen synthesis. A lack of vitamin C leads to scurvy, a well known scourge of

In addition to its antioxidant action in solution and its interaction with

Scheme 1.4: Ascorbic acid at different pH and L-ascorbyl-6-palmitate

1.5 Experimental approaches to elucidate antioxidant action

EPR is used in the investigation of radicals and their chemical reactions.

Fluorescence recovery after photobleaching (FRAP) employs fluorescently labeled

Closely related to antioxidant research is the investigation of the influence of

1.5.1 DBO as a fluorescent probe for antioxidants

the bridgehead carbon, is possible and has yielded an ever-growing family of

Scheme 1.5: The fluorazophore dye family.

The n,* singlet-excited state of DBO can be efficiently quenched by substances

1.5.1.1 Fluorazophore-L: A lipid-soluble DBO derivative

Scheme 1.6: Integration of Fluorazophore-L and -tocopherol in a lipid membrane

1.5.1.2 Synthesis of Fluorazophore-L

In step a) crotonaldehyde (1) reacts in a one-pot reaction with diethylamine in a

Scheme 1.7: Synthesis of the Fluorazophore-H

Scheme 1.8: Synthesis of Fluorazphore-L, 1-hydroxymethyl-DBO-palmitoyl ester

1.6 DBO and its derivatives as fluorescent probes in the

Scheme 1.9: Fluorazophore-L quenching by antioxidants in a liposome.

1.6.1 Quenching of Fluorazophore-L by vitamin C

1.6.2 Quenching of Fluorazophore-L by vitamin E

Figure 1.1: Diffusion in membrane models a) in an SDS-micelle (ideal micellar

As stated above, vitamin E is a collective term for a class of lipid-soluble