THE GLUTATHIONE THIYL RADICAL - Research Collection6052/eth-6052-02.pdf · her wenig strukturelle...

DISS. ETH NO. 20252

THE GLUTATHIONE THIYL RADICAL

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by

Dustin René Hofstetter

Dipl. Chem. ETH

born on 21st December 1977

citizen of Steinhof (SO)

accepted on the recommendation of

Prof. Dr. Willem H. Koppenol, examiner Prof. Dr. Ive Hermans, co-examiner

2012

Für all jene, welche an mich geglaubt haben, als ich es nicht mehr tat.

Allen voran meine Eltern, meine Grossmutter, mein Andreas

und mein Doktorvater.

Mit speziellem Dank für Rat und Tat an Reinhard Kissner, Thomas Nauser

und Heinz Rüegger(†), dessen Expertise kleinster Teil hierin bewahrt sei.

Edited, printed and bound by the author.

ISBN 978-3-033-03379-5

« Obélix, coupe trois parts de ce gâteau ! »

4

Table of Contents

Kurzfassung 6

Resumé 8

Summary 10

Abbreviations 12

1. Introduction 17 1.1 Thiols 18 1.2 Glutathione 19 1.3 The Biochemistry of Glutathione 20 1.4 The Glutathione Thiyl Radical 27 1.5 S–Nitrosoglutathione 33 1.6 Aim of this Work and Strategy 37

2. Materials, Methods and Instruments 39 2.1 Materials 40 2.2 Pulse Radiolysis 43 2.3 Hydrogen Exchange Sample Preparation and Measurement 46 2.4 Laser Flash Photolysis 48 2.5 Disulfide Synthesis 49 2.6 NMR Coalescence Measurement 50

3. Hydrogen Exchange Equilibria in Glutathione Radicals: Rate Constants 51

3.1 Introduction 52 3.2 Results 55 3.3 Discussion 60 3.4 Acknowledgement 65

4 Hydrogen Exchange Equilibria in Glutathione Radicals: Sites 67


5

5 The Glutathione Thiyl Radical Does not React with Nitrogen Monoxide 81


6 Synthesis of Disulfides from S–Nitrosothiols 89 6.1 Synthesis Letter 90 6.2 Acknowledgement 92

7 Coalescence of tert-Butyl-S–nitrosothiol 93 7.1 Introduction 94 7.2 Results 100 7.3 Discussion 108 7.4 Acknowledgement 112

8 General Conclusion and Perspectives 113

9 References 117

Curriculum Vitae 133

6

Kurzfassung

Glutathion (abgekürzt GSH) ist ein Stoff, der in vergleichsweise hohen Kon-

zentrationen im menschlichen Körper vorkommt und ist deshalb wichtig. Es

spielt eine wichtige Rolle bei Reaktionen, die das Redox-Gleichgewicht in der

Zelle erhalten. Es wurde schon für Aminosäuren und andere Peptide gezeigt,

dass Schwefelradikale intramolekular Wasserstoffatome von Kohlenstoff abs-

trahieren können. Um diesen Sachverhalt an Glutathion Schwefelradikalen

(GS•) zu untersuchen wählten wir zwei Methoden aus: Pulsradiolyse kombi-

niert mit UV-Vis, welches eine sehr hohe Zeitauflösung bietet, dafür aber e-

her wenig strukturelle Information, und NMR-Spektroskopie, welche zwar

eine sehr detaillierte räumliche Auflösung hat, aber keine saubere, eher direk-

te Produktion der gewünschten Radikale erlaubt.

Reduziert man oxidiertes Glutathion (GSSG) mit hydrierten Elektronen und

Wasserstoffatomen aus einer Pulsradiolyse, so ist die Umwandlung in das

Glutathion Radikalanion (GSSG•–) quantitativ. Dieses Radikalanion dissozi-

iert zu Glutathion Schwefelradikalen und Glutathion Thiolat (GS–), und das

dabei entstandene Schwefelradikal abstrahiert intramolekular Wasserstoff-

atome. Wir beobachten eine sequentielle Entwicklung von UV-

Absorptionssignalen, die auf die Bildung von sowohl !- und auch " -

Kohlenstoffradikalen hinweist. Aus den Experimenten bei pH 2 und pH 11.8

wurden die Geschwindigkeitskonstanten für die Hin- und die Rückreaktion

des Gesamtgleichgewichts zwischen Schwefel- und Kohlenstoffradikalen be-

stimmt: k! = 3#105 s–1 und k" = 7#105 s–1, und K = 0.4. Des weiteren konnte

mit Hilfe der Differenzen der Spuren bei 240 nm und bei 280 nm ermittelt

werden, dass das Verhältnis von !- zu "-Kohlenstoffradikalen überra-

schenderweise 1:3 ist. Die Bildung solcher Radikale kann zu irreversiblem

Schaden an den Proteinen führen, da sich Bindungen zwischen Kohlenstoff-

Atomen bilden können oder die Peptidhauptkette fragmentieren kann.

7

NMR-Experimente mit AAPH als Radikalinitiator und Propan-2-ol und Phe-

nylmethanol als Radikalketten-Verlängerer wurden benutzt um RS• aus cys-

teinhaltigen Peptiden RSH zu erzeugen. Mehrere gekoppelte Gleichgewichte

führen dazu, dass an Kohlenstoff gebundenes Protium durch Deuterium aus-

getauscht werden kann. Die Resultate schränken die möglichen Orte zwar ein,

lassen keinen eindeutigen Schluss zu, wo der Austausch stattfindet. Man er-

kennt dennoch, dass die Struktur und die Ionisation (pH-Wert) einen starken

Einfluss auf die Ergebnisse haben. Die einzige Tendenz, die sich ausmachen

lässt, ist die Verstärkung des D/H-Austausches wenn Stickstoffatome (oder

ein freies Elektronenpaar) in der Nähe des Thiylradikals vorhanden sind. Die

NMR-Experimente zeigen auch, das Glutathion in saurer Umgebung umla-

gert, die vorgeschlagene Struktur eines Thiolactons passt zu den erhaltenen

Daten und Reaktionsbedingungen.

Thiylradikale können eine Quelle für S–Nitrosoglutathion sein, da im selben

Kompartiment ebenfalls freies NO• vorkommt und eine simple Radikalkom-

bination das Produkt bilden würde. Experimente mit Laser Flash Photolyse

ergaben, dass die Geschwindigkeitskonstante für die Reaktion des Glutathion-

Thiylradikals mit NO• kleiner sein muss als 2.8 ± 0.6#107 M–1 s–1. Die Um-

wandlung des Schwefelradikals zum Kohlenstoffradikal bei 103 s–1 ist schnel-

ler als die Bildung von S–Nitrosoglutathion, wenn man physiologische Kon-

zentrationen von Stickstoffmonoxid voraussetzt. Es muss also andere Entste-

hungswege geben, die effizienter sind und damit auch relevanter.

Thiylradikale haben ein breites Spektrum an Reaktionsmöglichkeiten welche

von Struktur und Umgebung, z.B. dem pH-Wert, abhängen. Wir zeigen hier,

dass ihre Reaktion oft unvorhersehbar und kontraintuitiv ist. Nichtsdestotrotz

haben sie ein grosses Alterungspotential und deshalb ist es wichtig, ihre Reak-

tivität zu verstehen.

8

Résumé

Le glutathion est un composé dont le corps possède une concentration assez

haute comparé à d’autres constituants, et cela le rend important en vue des

processus cellulaires. Ce composé joue un rôle très important pour les

réactions responsables de la gestion de l’équilibre redox dans la cellule. La

possibilité d’enlèvement d’atomes d’hydrogène par des radicaux de soufre

(GS•) fût découverte déjà pour des acides aminés et d’autres peptides. Pour

investiguer si ces réactions sont aussi possibles pour le glutathion, nous

choisîmes deux méthodes : la radiolyse pulsée en combinaison avec UV-Vis,

qui a une résolution temporelle très grande mais qui apporte seulement peu

d’information sur la structure de l’espèce observée, et la spectroscopie RMN,

qui donne une résolution spatiale très fine mais manque d’une méthode nette

de production des radicaux souhaités.

La réduction de glutathion oxydé GSSG à son ion radical GSSG•– avec des

électrons hydratés et des atomes d’hydrogène produit par une radiolyse pulsée

se déroule quantitativement. Cet ion radical se désintègre en radicaux de

soufre et le thiolate de glutathion (GS–). Le radical de soufre qui se forme

dans ce processus est capable d’enlever des atomes d’hydrogène dans la

même molécule. Nous observons une séquence de signaux d’absorption dans

l’UV qui indique la formation de radicaux de carbone en positions ! et ". Les

expériences à pH 2 et pH 11.8 permirent la détermination des constantes de

vitesse de la réaction progresse (3#105 s–1), de la réaction régresse (7#105 s–1)

et la constante de l’équilibre (K = 0.4). En plus, les différences entre les

évolutions temporelles de l’absorption à 240 nm et 280 nm indiquèrent que le

rapport entre les radicaux de carbone en positions ! et " est 1 : 3, ce qui est

très surprenant. La formation de tels radicaux peut mener à la formation de

liaisons carbone-carbone ou à la fragmentation de la chaîne principale des

peptides et ainsi contribuer à des dégâts irréversibles auprès des protéines.

9

Dans les expériences RMN (résonance magnetique nucléaire) des peptides

contenant de la cystéine sont transformés en radicaux de soufre avec

AAPH comme source de radicaux et alcool isopropylique ou alcool

benzylique comme agents transporteurs des radicaux. Dans de telles

expériences des atomes de protium liés à des atomes de carbone peuvent être

échangés par des atomes de deutérium à cause de plusieurs équilibres

interdépendants. Les résultats ne permettent pas de localisation exacte des

sites d’échange, mais il est évident que la structure et l’ionisation (valeur pH)

influencent fortement les résultats. L’amplification de l’échange en présence

d’atomes d’azote (ou des doublets non liants) est la seule tendance qui se

manifeste. Le site préférentiel d’échange de glutathion est celui de la

glutamine " et $ à pH 2 et cysteine ! à pH 7. Les expériences montrent aussi

que glutathion se transforme très lentement en solution légèrement acide

(pH 3), la suggestion de structure est en accord avec les données obtenues et

les conditions de réaction.

Les radicaux de soufre peuvent être une source pour S–nitrosoglutathion,

parce qu’il y a du monoxyde d’azote NO• dans le même compartiment

cellulaire. Des expériences de photolyse « flash » de laser produisirent une

limite supérieure pour la constante de vitesse de la réaction entre le radical de

soufre de glutathion et le monoxyde d’azote : 2.8 ± 0.6#107 M–1 s–1. Si on

tient compte de la concentration physiologique de NO•, la transformation du

radical de soufre en radical de carbone est plus rapide (103 s–1) alors, il y a des

chemins de formation qui sont plus efficaces et, plus relevant.

Le Destin des radicaux de soufre est déterminé par une grande pluralité de

possibles réactions et il est dépendent de leur structure et de l’environnement.

Dans cette thèse je démontre que le comportement de ces radicaux est souvent

imprévisible et contre l’intuition. Tout de même, ils ont un grand potentiel de

vieillissement et c’est pourquoi il est impératif de comprendre leur réactivité.

10

Summary

Glutathione is an abundant and thus important compound in the human body.

It is involved in reactions that maintain the redox state of a cell. It has been

shown for other peptides with thiol groups, that their sulfur-centred radicals

abstract hydrogen atoms intramolecularly from carbon atoms. In order to in-

vestigate the behaviour of glutathione thiyl radicals in this respect two meth-

ods were chosen: pulse radiolysis combined with UV-Vis, which allows to

have a very good time resolution but limited structural information, and NMR

spectroscopy, which yields a very detailed structural information but deprives

of a clean and rather direct radical generation method.

The reduction of glutathione disulfide GSSG by hydrated electrons and hy-

drogen atoms from pulse radiolysis to form GSSG•– is quantitative. This radi-

cal anion dissociates into GS• and GS–, and the S-centred radical subsequently

abstracts a hydrogen intramolecularly. We observe sequential development of

UV absorbance signatures that indicate the formation of both !- and "-

carbon-centred radicals. From experiments performed at pH 2 and pH 11.8,

we determined forward and reverse rate constants for the overall equilibrium

between sulfur-centred and carbon-centred radicals: k! = 3#105 s–1 and

k" = 7#105 s–1, and K = 0.4. Furthermore, based on the differences between

the kinetics traces at 240 nm and 280 nm, we estimate that !- and "-carbon-

centred radicals are formed at a surprising ratio of 1:3. The formation of such

radicals could lead to irreversible damage in proteins via formation of carbon-

carbon bonds or backbone fragmentation.

NMR experiments with AAPH as a radical initiator and propan-2-ol and phe-

nylmethanol as radical propagators generated RS• from cysteine containing

peptides RSH. Several coupled equilibria lead to exchange of protium atoms

bound to carbon atoms by deuterium atoms. The results do not allow conclud-

ing on a predictable exchange pattern, rather structure and ionization

11

(pH values) have a strong influence on the results. The only feasible tendency

is the enhancement of D-/H-exchange in the presence of nitrogen (or a lone

electron pair) in a proximal position to the thiyl radical. In particular, gluta-

thione exchanges at pH 2 mostly at the glutamine " and $ positions, at pH 7

rather and strongly at the Cys ! position. Phenylalanyl-cysteinyl-glycine ex-

changes at pH 2 the more the closer the hydrogen atom is to the sulfur atom,

but Histidyl-cysteinyl-glycine shows a pronounced increase in exchange at

the His residue proximal to the nitrogen atom. NMR experiments also re-

vealed a re-arrangement of glutathione in acidic media, the suggested struc-

ture of a thiolactone fits the experimental data and reaction conditions.

Thiyl radicals could be a source of S–nitrosoglutathione as there could be free

NO• present in the same compartment and a simple radical combination

would lead to this product. Laser flash-photolysis experiments shows that the

rate constant for the reaction of the glutathione thiyl radical with oxidonitro-

gen(•) to give S–nitrosoglutathione is lower than 2.8 ± 0.6#107 M–1 s–1. The

conversion of the thiyl radical to its carbon-centred form at 103 s–1 exceeds the

formation of S–nitrosoglutathione when physiological concentrations of ox-

idonitrogen(•) are taken into account. Other pathways must be more efficient

and thus more relevant.

Thiyl radicals show a wide variety of possible reactions depending on struc-

ture and environment, e.g. the pH-value. Here we show that their reactions are

often not predictable and counter-intuitive, nevertheless they have a high po-

tential as a cause of ageing and therefore it is important to understand their

reactivity.

12

Abbreviations and Compound Names #including trivial names$ 2D-NMR two dimensional nuclear magnetic resonance spectroscopy AAPH !,!'-azodiisobutyramidin-dihydrochlorid Abs absorption units ADP adenosine diphosphate HAsc% ascorbate ATP adenosine triphosphate Br2

•% dibromide(•1%) Bu butyl c(X) concentration of X CDCl3 d-trichloromethane #deuterochloroform$ CO2

•% dioxidocarbonate(•1%) CO3

•% trioxidocarbonate(•3%) Cys cysteinyl residue d. doublet D2O 2H2O, d2-water DCl 2HCl, deuterium chloride

DHA dehydroascorbate e%aq

hydrated electron est estimate, estimated Et ethyl FAD flavin adenine dinucleotide G stem radical of glutathione without the thiol group G Gibbs free energy Glu glutamyl residue Gly glycyl residue GPx glutathione peroxidase {enzyme} [GS(H)SG]• glutathione disulfide radical GS• glutathione sulfur-centred radical, glutathione thiyl radical •GS glutathione carbon-centred radical GSH glutathione {reduced} -GSH glutathione structure as a part of a compound GSNO S–nitrosoglutathione

13

GSOO• glutathione thioperoxyl radical GSR glutathione reductase {enzyme} GSSG glutathione disulfide #oxidised glutathione$ GSSG•% glutathione disulfide radical anion Gy Gray {dose unit} H,H-COSY proton-proton correlation magnetic resonance spectroscopy H2O2 dioxidane #hydrogen peroxide$ HCO3

% hydrogen trioxidocarbonate #hydrogen carbonate$ hex. hexatuplet His-Cys-Gly histidyl-cysteinyl-glycine HNO hydrido-oxidonitrogen #nitroxyl$ HNO2 hydroxido-oxidonitrogen #nitrous acid$ H2NO2

+ dihydroxidonitrogen(1+) HO• oxidanyl(•) or hydridooxygen(•) #hydroxyl$ HOO• dioxidanyl(•) #hydroperoxyl$ HOONO (hydrido(dioxido))-oxidonitrogen #%&'()*nitrous acid$ HON=NOH diazenediol #hyponitrous acid$ HONO hydroxido-oxidonitrogen #nitrous acid$ i 2-methyl- structure as a part of a compound iso- 2-methyl- structure as a part of a compound J coupling frequency in Hz k rate constant of a chemical reaction K equilibrium constant of a chemical equilibrium reaction KSCN potassium nitridosulfidocarbonate(1%)

#potassium thiocyanate$ L Lewis-acid, ligand Lip- lipid structure as a part of a compound M mol · dm%3 (concentration unit) m. multiplet Me methyl meta- 1,3- (substituent positions in a benzene related structure) n nonbonding orbital N(CH3)4Cl tetramethylammonium chloride

14

N2O3 dinitrogentrioxide, N,N,N’-trioxidodiazane N3

• trinitrogen(•) #azide radical$ N3

•% trinitride(•1%) #azide radical anion$ Na3PO4 sodium tetraoxidophosphate(3%) #sodium phosphate$ NADPH adenine dinucleotide phosphate NaH2PO4 sodium dihydorxido-dioxidophosphate(3%)

#sodium dihydrogen phosphate$ Na2HPO4 sodium hydroxido-trioxidophosphate(3%)

#sodium hydrogen phosphate$ NaNO2 sodium dioxidonitrate(1%) #sodium nitrite$ Nd:YAG neodymium doped yttrium-aluminum-garnet neo- ((2,2-dimethyl)-ethyl)- structure as a part of a compound NMR nuclear magnetic resonance spectroscopy NO• oxidonitrogen(•) #nitric oxide$ NO+ oxidonitrogen(1+) #nitrosyl$ NO2

• dioxidonitrogen(•) or nitrogen dioxide O2

•% dioxide(•1%) #superoxide radical$ -O2

• dioxygen(•) group as a part of a compound -OOH hydroperoxide group as a part of a compound O2NNO2 N,N,N’,N’-tetraoxidodiazane ONOO• (dioxido)-oxidonitrogen(•) ONOO% (dioxido)-oxidonitrate(1%) #peroxynitrite$ ONOONO &-(dioxido)-di(oxidonitrogen) para- 1,4- (substituent positions in a benzene related structure) pD %log10 c(2H+(aq)) Pen penicillaminyl pH %log10 c(H+(aq)) Phe-Cys-Gly phenyl-cysteinyl-glycine Pi tetraoxidophosphate(3%) #inorganic phosphate$ %! %log10 ! Pr propyl Prot- or Protein-

polypeptide structure {protein} as a part of a compound

15

quint. quintuplet R universal gas constant R–N=N–R diazene structure as a part of a compound R, R’, R’’, … unspecified parts of a compound ROH compound containing a hydroxy group ROOH compound containing a hydroperoxide group RS• compound containing a sulfur radical RS+ compound containing a sulfylium group RS% compound containing a thiolate group RSeH compound containing a selenol group RSeOH compound containing a selenadioxide group RSNO compound containing a S–nitrosothiol group RSSR compound containing a disulfide group s. singlet (SCN)2

•% &-(disulfido)-di(nitrido)-dicarbonate(•1%) #dithiocyanate radical anion$

Sec selenocysteinyl residue -SeS- selenadisulfide group SOD superoxide dismutase {enzyme} t. triplet t' half-life T thermodynamic temperature tert 2,2-dimethyl- structure as a part of a compound Tyr tyrosyl residue TyrOH tyrosine UV-Vis photo spectrometry in the range from 200 – 1000 nm ! chemical shift relative to tetramethylsilane " molar absorbance per cm of absorbant # angle theta ( / (* bonding pi-orbital / anti-bonding pi-orbital ) / )* bonding sigma-orbital / anti-bonding sigma-orbital +XYZ Angle between the atoms X, Y and Z ,-./ Concentration of X

Chapter 1

Introduction

Chapter 1

18

1.1 Thiols

In biology, the chemical elements hydrogen, carbon, nitrogen and oxygen,

account for most of the organic compounds. In amino acids, sulfur and (to a

much smaller extent) selenium complete these building elements. Given their

abundance and functionality, amino acids belong to the most important class

of compounds in nature, and sulfur in the form of thiols (RSH), i.e. cysteine

(Cys), provides an important mechanistic and structural unit due to its capa-

bility of forming disulfide bridges by forming disulfides (RSSR).

2 RSH ! RSSR + 2 e% + 2 H⁺ (1.1)

The formation of disulfides involves a redox reaction and the formation of a

covalent bond, which offers many possibilities of interacting with the sur-

roundings. Mechanistically, this may be due to the unavailability of thiol

group in the disulfide or a change in redox state. Structurally, disulfide bridg-

es allow proteins and peptides to achieve their final tertiary structure, e.g. in-

sulin, antibodies, or lipase. Among the thiols, glutathione (Fig. 1.1) is most

the abundant one; it is commercially available at reasonable price, and there-

fore most suitable as a model compound.

Introduction

19

1.2 Glutathione

The glutathione molecule is a tripeptide consisting of a $-linked glutamate

(Glu), followed by a cysteine and terminated by a glycine (Gly), systematical-

ly named as $-L-glutamyl-L-cysteinylglycine. Glutathione is often abbreviat-

ed as GSH, representing its initial letter “G” and its most important functional

group, the thiol. Although glutathione has a most important role in metabo-

lism, it cannot be taken up by nutrition: enzymes of digestion would immedi-

ately cleave the peptide into its individual amino acids. Most mammalian cells

can synthesise glutathione on their own.

Figure 1.1: The idealised structure of glutathione

The dimeric form of glutathione consists of two glutathione units linked by a

disulfide bridge. It is commonly abbreviated by GSSG, in analogy to glutathi-

one. The formation of the disulfide bridge (–SS–) with concomitant loss of

two hydrons (H+) is an oxidative process.

Glutathione has several acidic groups, and therefore several pK values:

pK (Glu, -COOH): 2.1 [1] pK (Gly, -COOH): 3.5 [1]

pK (Cys, -SH): 9.2 [2] pK (Glu, -NH3+): 9.5 [2]

At physiological pH, i.e. pH 7.2 – 7.4, the carboxylic acid groups are depro-

tonated and most (>98%) of the thiol and amine groups will be protonated.

The degree of protonation is important for the reactivity of glutathione, par-

ticularly of the thiol.

Chapter 1

20

1.3 The Biochemistry of Glutathione

Figure 1.4 (p. 25) gives an overview of the most important reactions in a

physiological environment. The numbers in bold italic print in this chapter

(e.g. 42) refer to the reactions drawn in that Figure.

The biochemical formation of glutathione consists of two steps: the formation

of $-glutamyl-cysteine with $-glutamyl-cysteine synthease 1 and subsequent

addition of glycine by glutathione synthease 2. Glutathione is very abundant

in the cytosol, where it can be found in concentrations of approximately

10–3 M [3]. This relatively high concentration, i.e. availability, may be the

reason, why glutathione took over multiple roles within the cell regulation

system [4]:

It can act as a detoxifying agent: its nucleophilic properties allow it to be con-

jugated with toxic species, often via the intermediacy of GS-S-transferases.

The products of these conjugations are often mercapturic acids. The cell has

mechanisms in place that transport these glutathione conjugates actively out

of the cell.

Glutathione can also act as a modulator by forming a disulfide bridge to cys-

teine carrying proteins, also summarised as “protein S-glutathionylation”. A

wide range of membrane-bound and cytosolic proteins including a variety of

proteins involved in cell apoptosis seem to undergo “S-glutathionylation”.

But it is important not only in terms of concentration: the reduced and oxi-

dised forms of glutathione have electrode potentials that makes them conven-

iently strong reducing (GSH) and oxidising (GSSG) agents. These are formed

in tisues not by accident (e.g. in contrast to partially reduced oxygen species),

but by intent:

RS• + e% !/01% (1.2)

Introduction

21

The standard electrode potential of reaction 1.2 is approximately +0.9 V [5].

The formation of the disulfide from the thiol has an electrode potential of ap-

proximately +0.27 V [6] :

GSSG + 2 H+ + 2 e% ! 2 GSH (1.3)

The abundance of these two glutathione forms and their electrode potentials

makes them sufficient a pool for balancing the redox state of the cell, i.e. to be

able to respond to oxidative stress.

The electrode potential of the GSSG/2 GSH couple, reaction 1.3, is rather

low, so that even ascorbate (HAsc%) can be restored from dehydroascorbate

(DHA), namely by dehydroascorbate reductase (E°’(DHA/Asc2%) = %0.08 V

[7]), that again turns two glutathione units into glutathione disulfide 11. Glu-

tathione can also restore (other) disulfides to the according thiols by thiol

transferase that also converts two glutathione units into glutathione disulfide

12. Glutathione peroxidase [8] converts dioxidane (H2O2) and lipid peroxides

(Lip-OOH) to water and hydroxy lipid compounds (Lip-OH) 13.

Glutathione peroxidase is one of the rare enzymes that contain selenium in

the form of selenocysteine (Sec). The catalytic cycle consists of three steps

(Fig. 1.2):

1. The peroxide (ROOH) oxidises the selenol group (enzyme-SeH) in the

protein to a selenadioxide group (enzyme-SeOH) and is itself reduced

to an alcohol (ROH).

2. The conjugation of the selenadioxide group to a glutathione unit reduc-

es yields a mixed selenadisulfide (enzyme-SeS-G) and releases a water

molecule.

3. The selenol in the enzyme recovers by another glutathione unit cou-

pling to the first glutathione unit and reducing the mixed selenadisul-

fide.

Chapter 1

22

The use of selenium in physiology is very peculiar and seems counter intui-

tive, because the mechanism given above does exist with ordinary cysteine in

the place of the selenocysteine in comparable enzymes of other species and

the biochemical processing of selenocysteine is rather complicated and in-

volves the alternative use of a DNA STOP codon [9]. Also the use of seleno-

cysteine brings along the unfavourable need to cover selenium as an addition-

al nutrient.

Figure 1.2: The catalytic cycle of glutathione peroxidase.

The answer might lie in the broader specificity of the selenoenzyme compared

to the thioenzyme [9; 10]: the selenol group is deprotonated at lower pH val-

ues, which increases the reactivity in nucleophilic substitutions. What makes

the substitution of sulfur by selenium even more important are the different

electrode potentials of the selenium compounds: these potentials are about

half of the according values for sulfur compounds. This is why the seleno-

enzyme is more reactive [11]. The coupling of two electrode potentials allows

also the crucial combination of one- and two-electron transfer reactions.

Eventually, fast enzyme rates are more advantageous than the dependency of

selenium and the management of its intake.

drawing: D. Hofstetter

Introduction

23

Figu

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Chapter 1

24

Although several pathways leading from glutathione to glutathione disulfide

have been published, they are by no means exhaustive. As a result, there is no

overview established of all the paths leading from glutathione to glutathione

disulfide. Finally, all the glutathione disulfide generated by such processes is

then recycled to glutathione by glutathione reductase 14 which consumes one

unit of nicotinamide adenine dinucleotide phosphate (NADPH) per unit gluta-

thione recycled (Fig. 1.3) [12]. Nicotinamide adenine dinucleotide phosphate

is one of two important carriers for reduction equivalents (for two-electron

reactions) next to flavin adenine dinucleotide FAD (for one-electron reac-

tions). The latter is used in the same process to stock one reduction equivalent

for the two-step reduction of glutathione disulfide.

Whereas glutathione peroxidase is optimised for speed and range of substrate,

glutathione reductase has only one specific substrate: glutathione disulfide.

And this substrate is non-toxic in contrary to the peroxide substrates for gluta-

thione peroxidase. This matches the fact that the cell stocks most of the total

glutathione in the form of glutathione (reduced) and is immediately available

for detoxification of peroxide species. This process eventually yields glutathi-

one disulfide, which is subsequently continuously “recycled” by reduction to

glutathione.

One of the most interesting derivatives of glutathione is S–nitrosoglutathione

(GSNO), where the oxidonitrogen(1+) (NO+) replaces the hydron (H+) at the

sulfur atom:

GS-H + NO+ GS-NO + H+ (1.4)

The exact way of biosynthesis 21 lies still in the dark and is subject to consid-

erable scientific dispute. In consequence, the intended or speculated physio-

logical function of this compound suffers from the same. The photolytic de-

cay to glutathione disulfide and oxidonitrogen(•) (NO•) 22 and the transfer of

the oxidonitrogen(1+) group from one thiol to another 24 are both established.

Introduction

25

Figu

re 1

.4: T

he m

ost i

mpo

rtant

reac

tions

for g

luta

thio

ne in

a p

hysi

olog

ical

env

ironm

ent:

reac

tions

1 a

nd 2

sho

w

the

bios

ynth

esis

, rea

ctio

ns 11

– 15

rep

rese

nt th

e m

ost i

mpo

rtant

red

ox r

egul

atio

n re

actio

ns (

HA

sc! =

asc

orba

te,

DH

A =

deh

ydro

asco

rbat

e, L

ip =

Lip

id,

Pro

t = P

rote

in), 21

– 24

are

the

reac

tions

tha

t in

volv

e S

–nitr

oso

com

-po

unds

, 31

– 39

are

the

reac

tions

of

sulfu

r- a

nd o

xyge

n-ce

ntre

d ra

dica

ls,

that

can

occ

ur,

reac

tions

41

and 42

de

al w

ith c

arbo

n-ce

ntre

d ra

dica

ls. C

oupl

ing

reac

tions

to o

ther

com

poun

ds (“

glut

athi

onyl

atio

ns”)

are

om

itted

.

draw

ing:

D. H

ofst

ette

r

Chapter 1

26

The possibility of direct formation of S–nitrosoglutathione form the thiyl rad-

ical (GS•) and oxidonitrogen(•) 23 has been investigated in this work. Further

details on S–nitrosoglutathione can be found in subchapter 1.5.

Glutathione is prone to hydrogen atom abstraction by species that contain par-

tially reduced oxygen or partially oxidised nitrogen atoms 31. In consequence,

glutathione is transformed in to the glutathione thiyl radical; and it is just this

glutathione thiyl radical that can either smoothly react with one another of its

own species to yield glutathione disulfide 32 or give rise to a multiplicity of

onward reactions 33 – 42. Further details on the glutathione thiyl radical can

be found in the subchapter 1.4 here after.

In addition to the aspects presented here, further biochemical implications

such as glutathione transport, protein folding, radiation protection and many

more are subject to current research [13].

Introduction

27

1.4 The Glutathione Thiyl Radical

There are many species and classes of radicals that can react with glutathione

in the so-called repair process. To date, it is assumed that this reaction leads

almost exclusively [13] to the glutathione sulfur-centred radical, also named

glutathione thiyl radical. Other reaction types are often summarised, omitted,

or neglected in research, because they do not yield the oxidised form of gluta-

thione that can be regenerated. The fate of this radical is rather complex, de-

pending on equilibria represented as process 37 (Fig. 1.4), which, in turn, de-

pends on the pH value [14]:

GS• + GSH 2/[GS(H)SG]• (1.5)

[GS(H)SG]• 2/[GSSG] •% + H+ (1.6)

GS• + GS% 2 [GSSG] •% (1.7)

At first, in the early 1980 decade, C-centred radicals were attributed to the

unselective reaction of glutathione with oxidanyl(•) radical (HO•) [15; 16] in

contrary to the dibromide(•1% ) radical (Br2•%) [15], which seemed to selec-

tively abstract hydrogen atoms from the sulfur atom:

GSH + HO• 2 GS• + H2O (1.8)

GSH + HO• 2 •GS + H2O (1.9)

GSH + Br2•% 2 GS• + 2 Br% + H+ (1.10)

Approximately ten years later it was found that the thiyl radical shows an

even more complex reactivity: it undergoes an internal transformation from an

S-centred radical to a C-centred radical [17]:

Chapter 1

28

(1.11)

Figure 1.5: The equilibrium 1.11 of S- and C-centred glutathi-one radicals. Impotantly, the stereochemistry may not be re-tained upon the back reaction.

Initially, it was regarded as a forward reaction only (1.11), the according hy-

droxide dependent rate constant was reported as k'1.11* = 103 s%1 at pH 7, with

an overall constant of k1.11* = 5 109 M%1 s%1. Only later [18], this process was

found to be an equilibrium reaction, although the backward constant k1.11+

could not be determined. More than ten years later, the rate constants for simi-

lar proteins were determined to k1.11* = 0.1–1 105 s%1 and k1.11+ = 1–9 105 s%1

at pH 4 [19].

The experimental generation of glutathione thiyl radicals is manifold. The use

of highly oxidising radicals, such as oxidanyl(•) [17; 20], dibromide(•1%)

[15], or trinitrogen(•) (N3•)[21] is well established. Photolysis is a further way

of generating the desired radicals with a high yield of thiyl radicals [22] di-

rectly. Recent experiments suggest, that the photolysis of disulfide bonds

might not be of homolytic nature but produce thiolate (RS%) and sulfylium

(RS+) species [23], even in symmetric thiols. Furthermore, there are indirect

ways to the thiyl radical, either by reducing glutathione disulfide by hydrated

electrons (e%aq) [18; 24], hydrogen atoms [25] or alternatively make use of

secondary alcohols (2-propanol, benzyl alcohol) as a mediator between radi-

cals of thermal decay and glutathione [26].

RN=NR 2 R• + N2 (1.12)

R• + R2CH(OH) ! RH + R2C•(OH) (1.13)

R2C•(OH) + GSH R2CH(OH) + GS• (1.14)

Introduction

29

Past experiments [15] show, that there are even faster reactions of the thiyl

radical can be observed, although they were not attributed to internal trans-

formations but to a reaction with hydroxyl, which in turn was rejected [17]

but not further investigated. This matter was not resoved.

The generation of glutathione thiyl radicals in a physiological environment

are somewhat simpler but not less interesting: the glutathione thiyl radical

forms, when oxidanyl(•) [27], oxidonitrogen(•) [28], trioxidocarbonate(•1%)

(CO3•%) [29] or membrane lipid radicals (Lip-OO•, Lip-O•) [30] react with glu-

tathione in a rather simple intermolecular hydrogen abstraction reaction 31.

These and other compounds that react in the same manner are transformed

into less oxidative compounds that carry also less potential for irreversible

cell damage. The direct reaction with another radical of its kind 32 is only

way to “dispose” of a glutathione thiyl radical without generation of further

radicals; the chances for that are relatively small compared to the number of

alternative targets that are available in a cell.

In contrast to the radicals above, the reaction of glutathione with dioxide(•1%)

(O2•%) is rather slow k1.15 , 200 M%1 s%1[31].

O2•% + GSH + H+ * GS• + H2O2 (1.15)

At average physiological concentrations dioxide(•1%) reacts mostly with su-

peroxide dismutase with a rather high rate constant of 2.3#109 M%1 s%1 [4; 32]:

2 O2•% + 2 H+

!!superoxide dismutase!! O2 + H2O2 (1.16)

This also means, that the redox balance of the cell is not strongly influenced

by escaping dioxide(•1%): superoxide dismutase transforms dioxide(•1%) into

dioxidane and dioxygen without the need for redox equivalents. The same

holds for catalase, an enzyme that converts dioxidane to water and dioxygen.

Although catalase is one of the most efficient proteins [33], some dioxidane

Chapter 1

30

will take the route via glutathione peroxidase 13, which then indeed would

require glutathione as reduction equivalents.

Dioxygen reacts readily with glutathione thiyl radicals to glutathione thioper-

oxyl radical (GSOO•) as the product 33. It reacts further with one 34 or two

equivalents 35 – 36 of glutathione to yield glutathione disulfide and dioxida-

nyl(•) radicals or dioxidane, respectively. Further dioxidane is the product of

superoxide dismutase 39 that consumes dioxide(•1%) from the reaction of di-

oxygen and the glutathione disulfide radical anion (GSSG•%) 38, which is a

direct product from a glutathione thiyl radical that reacts with glutathione. As

much as glutathione thiyl radicals can be the source for dioxidane, glutathione

can act as a sink for dioxidane in glutathione peroxidase 12 [8]. So the gener-

ation of one glutathione thiyl radical that reacts with dioxygen is not only the

loss of one, but up to five reduction equivalents in the form of glutathione,

that are consumed at restoring, with high chances of another glutathione thiyl

radical as a product at the end of reaction 35.

The glutathione thiyl radical can also be the acceptor in an intermolecular

hydrogen abstraction with other amino acids, peptides or proteins as donors

(not shown in Fig 1.4).

Protein-H + GS• 2 Protein• + GSH (1.17)

The rate constants for typical protein radicals for forward and reverse reac-

tions are approximately equal (vide infra). This means that a hydrogen trans-

fer from the protein to glutathione is probable, which is important: it shows,

that the glutathione thiyl radical is not a relatively “stable” and thus harmless

radical, but can pass the reactivity of the unpaired electron on to more com-

plex and therefore possibly more valuable compounds such as proteins.

According to the principle of microscopic reversibility proteins with unpaired

electrons, so called “damaged proteins”, Protein•, (from reactions with species

that contain partially reduced oxygen atoms or partially oxidised nitrogen at-

Introduction

31

oms), could abstract sulfur-bound hydrogen atoms from glutathione and be

“repaired” to the original state (Protein–H). Still, the reaction rate at concen-

trations given at physiological conditions is not sufficient to outcompete

ascorbate: damaged proteins undergo reaction with ascorbate faster than with

glutathione [34]: equilibrium 1.18 is the reversed equilibrium 1.17 with a ty-

rosine residue of a protein. It is thermodynamically well balanced, i.e.

K1.18 , 1.

TyrO• + GSH TyrOH + GS• (1.18)

An efficient repair would not seem probable under these circumstances, but

there are more possible reactions for glutathione thiyl radicals than for tyro-

sine radicals (TyrO•). Therefore a sink of GS• could make a “repair” reaction

(1.18) is kinetically favoured, but only in the absence of any ascorbate, which

reacts under physiological conditions approximately 200 times faster than

glutathione to form the ascorbyl radical:

TyrO• + AscH TyrOH + Asc•% (1.19)

In turn, the ascorbyl radical can transform into dehydroascorbate (DHA),

which is restored by glutathione and the according enzyme 14.

Alternatively, the glutathione thiyl radical can undergo an intramolecular hy-

drogen abstraction and turn from a sulfur-centred radical to a carbon-centred

radical 41. This might occur by a 1,2-H-shift or a 1,3-H-shift, but also hydro-

gen atoms located further away from the sulfur atom are subject to this pro-

cess. This reaction contains the very important implication that carbon-

centred radicals are formed and any consequent carbon-carbon bond for-

mation 42 is quasi permanent and represents an irreparable damage to the

compound concerned. In contrast, sulfur-sulfur bonds from sulfur-centred rad-

icals can be split and restored to thiols relatively easily. In order to understand

Chapter 1

32

more of these processes involving sulfur- and carbon-centred radicals and

their nature, this reaction was investigated in this work.

Introduction

33

1.5 S–Nitrosoglutathione

S–nitrosoglutathione is glutathione molecule modified at the sulfur atom,

where a oxidonitrogen(1+) (NO+) group replaces the proton attached to the

sulfur. S–nitrosoglutathione is commonly abbreviated by GSNO [35].

The chemical synthesis of S–nitrosoglutathione is easy and follows known

routes for nitrosations by hydrogen dioxidonitrate [36]:

HCl + NO2% ! HONO + Cl% (1.20)

2 HONO ! N2O3 + H2O (1.21)

N2O3 + GSH ! GSNO + HNO2 (1.22)

The reaction could also occur via dihydroxidonitrogen(+) (H2NO2+), which

essentially is an adduct of oxidonitrogen(1+) to a water molecule (reac-

tion 1.23), instead via reactions 1.21 and 1.22 [37].

H+ + HONO 2 H2NO2+ 2 H2O + NO+ (1.23)

The rate law of v = k·c(HONO)·c(H3O+)·c(GSH) and the reaction rates found

for S–nitrosothiol formation do not allow distinguishing between these path-

ways. Two further ways of S–nitrosothiol preparation include the bubbling of

oxidonitrogen(•) through a aerated solution of the desired thiol in methanol

for 3 h and alternatively the addition of methylbenzene (C6H5–CH3) in order

to form two phases, the organic of which contains the desired product [38].

More important is the biosynthetic route to S–nitrosoglutathione. It is of ma-

jor importance to understanding its involvement in metabolism and its func-

tions. Nevertheless, this is still being explored and there are several hypothe-

ses for 21:

One suggested route follows the chemical synthesis very closely; it involves

nitrosation by N2O3 an auto-oxidation product oxidonitrogen(•), a vasodilator

produced in the endothelium [39]:

Chapter 1

34

2 NO• + O2 * 2 NO2 (1.24)

NO• + NO2• 2 N2O3 (1.25)

N2O3 + GSH ! GSNO + HNO2 (1.22)

It can be seen, that for the generation of one S–nitrosoglutathione molecule

three oxidonitrogen(•) molecules are needed. In view of the low physiological

concentration of oxidonitrogen(•) of approximately 10–7 M [40], this does not

seem a very probable reaction. Indeed, the precursor molecules are also very

reactive and can react with a number of possible targets before reacting with

glutathione.

The direct reaction of oxidonitrogen(•) and glutathione thiyl radicals is a se-

cond speculative route, whereby the glutathione thiyl radical from repair reac-

tions (vide supra) reacts directly to S–nitrosoglutathione.

GS• + NO• ! GSNO (1.26)

There are aspects to this proposition that make it also unlikely. Whereas ox-

idonitrogen(•) is produced in the endothelium (blood vessels) and in neuronal

tissue [41], the thiyl radicals are produced at sites where considerable cell

respiration takes place, while S–nitrosoglutathione is found in the plasma and

therefore affects the entire body [42].

S–Nitrosoglutathione can also be the result of a reaction of transition metal

complexes (L%Fe, e.g. iron(II), copper(II)) and oxidonitrogen(•) (reactions

1.27–1.30). Transition metal complexes also catalyse trans-nitrosation reac-

tions between thiols and S–nitrosothiols [43; 44; 45; 46].

L-FeIII + NO• L-FeIII-(NO) (1.27)

L-FeIII-(NO) L-FeII-(NO+) (1.28)

L-FeII-(NO+) + RSH !/L-FeIII + RSNO + H+ (1.29)

RSNO + GSH RSH + GSNO (1.30)

Introduction

35

However, this seems only a minor route in oxidonitrogen(•) oxidation by lig-

and bound iron and might not account for the amounts of S–nitrosoglutathione

found [46; 47].

There are recent speculations that deals with possible intermediates of the ox-

idation of oxidonitrogen(•) by dioxygen [48]

NO• + O2 ONOO• (1.31)

ONOO•+ NO• ONOONO (1.32)

ONOONO O2NNO2 (1.33)

O2NNO2 2 NO2 (1.34)

The intermediate (dioxido)-oxidonitrogen(•) (ONOO•) is a possible nitrosat-

ing (or nitrosylating) agent that could react with glutathione or its thiyl radical

to yield S–nitrosoglutathione. Those radicals would be available in a steady

state in very low concentrations only. Whether the total turnover could ac-

count for the S–nitrosoglutathione concentrations found is yet to be elaborat-

ed.

The fate of S–nitrosoglutathione is simpler than its speculative genesis. It can

release oxidonitrogen(•) upon reduction (vide infra), photolysis [49] (1.22) or

it can exchange the nitroso group with any other thiol by (re-)trans-nitrosation

(-1.30). Thermolysis was also discussed as a way for the decay [50], but there

is evidence that this process is irrelevant [51].

!"GSNO !!!!!!

GSSG + !"NO• (1.35)

S–nitrosoglutathione also reacts readily with ascorbic acid or ascorbate pre-

sent at approximately 50 &M or higher in the human body [52; 53; 54]:

GSNO + Asc2% ! GS% + Asc•%+ NO• (1.36)

Chapter 1

36

Also, ascorbate can reduce Cu2+, which gives rise to a cyclic reaction cata-

lysed by copper(II)-ions (1.37), e.g. conceivably from Cu,Zn-Superoxide dis-

mutase, or by other heavy metal ions that can be reduced by ascorbate [37]:

Cu2+ + Asc2% ! Cu+ + Asc•% (1.38)

Cu+ + GSNO ! Cu2+ + GS% + NO• (1.39)

Small concentrations as 10%4 M ascorbate are already effective with cop-

per(II)-ions present, the uncatalysed reaction requires higher concentrations of

ascorbate of approximately 10%2 M. The rate constant is pH dependent, be-

cause the di-anion of the ascorbic acid accounts for most of the reaction, k1.36

amounts to approximately 10%2 M%1 s%1 at pH 7 [37].

The biological role of S–nitrosoglutathione is still being discussed. Possible

functions are a sink, a transporter, or storage for oxidonitrogen(•), which fits

very much with the mechanisms of generation, trans-nitrosation and destruc-

tion presented above. Considering its capability of releasing oxidonitrogen(•),

the scope of possible interactions becomes even larger. Thus, it is very im-

portant, which way will establish as the correct one in biosynthesis in order to

gain more insight in the role of S–nitrosoglutathione.

Introduction

37

1.6 Aim of this work and strategy

The aim of this work is to understand the behaviour of thiyl radicals. This in-

cludes primarily the behaviour immediately after formation, i.e. elucidating

H-abstraction mechanisms. Furthermore the behaviour towards the oxido-

nitrogen(•) molecule is investigated, in order to contribute to the knowledge

of S–nitrosoglutathione formation.

Since time resolution and structural information are both crucial in this task,

pulse radiolysis with UV-Vis detection (Chapter 3) and laser flash photolysis

(Chapter 5) were chosen to satisfy the time resolution. NMR spectroscopy

provided structural information (Chapter 4). The thermal decomposition of S–

nitroso compounds opened a facile route to disulfides (Chapter 6) and vibra-

tional phenomena were investigated with UV-Vis and NMR temperature

range experiments (Chapter 7).

Although the methods are established, the adaption to the planned research is

a major point of this work and careful data analysis is the other.

Chapter 2

Materials, Methods and Instruments

This chapter unifies the Methods and Instrumental sections of the chap-

ters and publications thereafter.

Chapter 2

40

2.1 Materials

All chemicals were used as delivered. Millipore Milli-Q purified water

(18.2 M-) was used throughout all experiment unless described differently.

Compound Full systematic name

Quality Company Place

L-Cysteine, Cys 99.5% Fluka Buchs, Switzerland

Cysteinyl-glycine, Cys-Gly 98% Bachem Bubendorf, Switzerland

Glutathione, GSH 97% Acros Bachem

Geel, Belgium Bubendorf, Switzerland

Glutathione disulfide, GSSG 98% Acros AppliChem Sigma

Geel, Belgium Darmstadt, Germany St. Louis, MO, USA

Phenyl-cysteinyl-glycine, Phe-Cys-Gly

96% Peptide 2.0 Chantilly, VA, USA

Histidyl-cysteinyl-glycine, His-Cys-Gly

97% Peptide 2.0 Chantilly, VA, USA

Deuterium oxide, D2O 99.96% Cambridge Iso-tope Laborotories Inc.

Andover, MA, USA

Deuterium chloride 35% in D2O, DCl

99.5% 2H

Cambridge Iso-tope Laborotories Inc.

Andover, MA, USA


41

Sodium Phosphate, Na3PO4 96% Aldrich St. Louis, MO, USA

d6-Isopropanol 99% 2H Aldrich St. Louis, MO, USA

!,!'-Azodiisobutyramidin-dihydrochlorid, AAPH

98% Fluka Buchs, Switzerland

Tetramethylammonium chloride, N(CH3)4Cl


Benzyl alcohol 99.8% Riedel-de Haën Seelze, Germany

tert-Butanol recryst. Merck Darmstadt, Germany

Potassium thiocyanate, KSCN


Sulfuric acid, H2SO4 >95% Merck Darmstadt, Germany

Potassium Hydroxide, KOH p.a. Brenntag Schweizerhall

Basel, Switzerland

Sodium dihydrogen phos-phate dihydrate, NaH2PO4·2 H2O

p.a. Fluka Buchs, Switzerland

Sodium hydrogen phosphate dodecahydrate, Na2HPO4·12 H2O

p.a. Fluka Buchs, Switzerland

Argon 5.0, Ar 99.999% Linde Höllriegels-kreuth, Germany

Nitric oxide 2.0, NO• 99.5% Linde Höllriegels-kreuth, Germany

Dodecanethiole C12H25SH


Butanethiole C4H9SH 99% Fluka Buchs, Switzerland

Chapter 2

42

1,1-dimethyl-ethanthiole C4H9SH


2,2-dimethyl-propanethiole C5H11SH


Table 2.1: List of the materials used including their purities and

origin.

All S–nitroso compounds were prepared according to the literature [36] and

stored at -18°C.

All activities of the species in solution were assumed to be equal to their con-

centrations unless mentioned differently.


43

2.2 Pulse radiolysis

Pulse radiolysis is the direct introduction of electrons into a solution via the

release of a short electron beam, the “pulse”. This electron pulse, in case of a

Febetron accelerator, is the product of a Marx-Generator type high voltage

generator containing 80 modules that allow trigger voltages up to 35 kV. Each

module contains a capacitor and is charged in a parallel circuit. (Fig. 2.1 A).

Triggered ionisation (i.e. an induced spark) releases a cascade of capacitor

discharges in a series circuit that add all the voltage differences of the capaci-

tors (Fig. 2.1 B).

Figure 2.1: Circuit drawing (with American standard circuit symbols) of the two stages of a Marx-Generator, grey parts of the circuit are inactive. A: The modules containing capacitors are charged in a parallel circuit. B: The capacitors of the mod-ules discharge in a series circuit and summing their voltages. (Modified from a source in public domain: http://en.wikipedia.org/wiki/File:Marx_Generator.svg)

Chapter 2

44

The irradiation of aqueous solutions yields primary radicals—the hydrated

electron, the hydrogen atom, and oxidanyl(•)—the dose dependent concentra-

tions of which are known [55]. The chemical yield of primary products

(Eqn. 2.1) is described with the G-Value, the number of molecules formed per

100 eV absorbed:

H2O(l) 2.6 e–aq, 0.6 H•, 2.7 HO•, 2.6 H+, 0.7 H2O2, 0.45 H2 (2.1)

In order to create uniform reaction conditions, the solution contains next to

the target molecules also compounds that react very rapidly with the primary

products to uniformly reducing species (e%aq, CO2•%, O2

•%) or uniformly oxidis-

ing species (Br2•%, HO•, N3

•%).

All work was done in glassware cleaned by imersion in concentrated nitric

acid followed by rinsing with pure water, and de-aerated glassware by at least

three-fold evacuation with subsequent shaking under argon at atmospheric

pressure. The solution was transferred via a Gastight® syringe from Hamilton

(Bonaduz, Switzerland) to a syringe pump for sample delivery. Each sample

was irradiated and analysed only once.

A Febetron 705 accelerator (Titan Systems Corp., San Leandro, CA, USA)

generates electron pulses of 50 ns duration and energies of 2.0 MeV. The dose

is adjusted with aluminum apertures, to 7–140 Gy/pulse. A xenon arc lamp

(75 W, Hamamatsu, Schüpfen, Switzerland) in combination with a R928 pho-

tomultiplier (also Hamamatsu), a DHPCA-200 amplifier (Femto Messtechnik

GmbH, Berlin, Germany) and a DL7100 digital storage oscilloscope (Yoko-

gawa Electric Corporation, T.ky., Japan) permits absorption changes versus

time (i.e. kinetic traces) to be recorded of the solutions in the custom made

quartz cell (Hellma GmbH & Co KG, Müllheim, Germany) equipped with

air-tight tubing for automatic sample refill (this setup has been described in

more detail in [11]). All experiments were conducted at 295 K.


45

The dosimetry was determined by irradiation of potassium nitridosulfidocar-

bonate(1%) (KSCN) solutions [56]: oxidanyl(•) oxidises the nitridosulfidocar-

bonate (1%) ion to the &-(disulfido)-di(nitrido)-dicarbonate(•1%) ion

((SCN)2•%) with an absorbance of 7.6#103 M%1 cm%1 at 472 nm, according to:

2 SCN% + HO• ! (SCN)2•% + HO% (2.2)

All solutions were prepared with 1 M tert-butanol to scavenge oxidanyl(•)

[57] (k2.3 = 6.0#108 M–1 s–1), with production of the relatively innocuous 2-

hydroxy-2-methylpropyl radical:

tert-BuOH + HO• !/•CH2(CH3)2C(OH) + 2 H2O (2.3)

Although the second-order rate constant for the reaction of glutathione disul-

fide with oxidanyl(•) is two orders of magnitude higher (9#109 M–1 s–1) [27],

the product of rate constant and concentration (1 mM), 9#106 s–1, is negligible

compared to that for the reaction of 1 M tert-butanol with oxidanyl(•),

6#108 s–1. Thus, the reactions we observe are those of hydrated electrons and

hydrogen atoms, which react with glutathione disulfide to form the glutathi-

one disulfide radical anion and its conjugate acid the glutathione disulfide

radical (GS(H)SG•) respectively [25; 18] with rate constants of

k2.4 = 3#109 M–1 s–1 and k2.5 = 9#109 M–1 s–1:

e–aq, + GSSG * GSSG•– (2.4)

H• + GSSG * GS(H)SG• (2.5)

Pulse radiolysis data was obtained as data points with two dimensions: time

and relative absorption difference. Only data points obtained after 500 ns

were evaluated; earlier time-points were neglected because the 50 ns pulse of

electrons created lingering electromagnetic disturbances. Kinetics curves were

smoothed with a digital low-pass filter, and the data were fit with a least

squares algorithm.

Chapter 2

46

2.3 Hydrogen Exchange Sample Preparation

and Measurement

The pD of solutions in deuterated water (D2O) was low (pD 1 or pD 2) or

neutral (pD 7.4); the solutions were de-aerated by at least three-fold evacua-

tion with subsequent shaking under argon at atmospheric pressure, and then

transferred to a glove box with an inert atmosphere. Cysteine, cysteinyl-

glycine (Cys-Gly), glutathione, histidyl-cysteinyl-glycine (His-Cys-Gly) and

phenylalanyl-cysteinyl-glycine (Phe-Cys-Gly) were used as target amino acid

or peptides. The solutions contained 2 mM cysteine or peptide, the radical

initiator !,!'-azodiisobutyramidin-dihydrochlorid (AAPH, 20 mM) and a rad-

ical propagator (20 mM benzyl alcohol for Cys, Cys-Gly and GSH and

20 mM d6-isopropanol for His-Cys-Gly and Phe-Cys-Gly). Tetrame-

thylammonium chloride (N(CH3)4Cl, 0.2 mM) was added to solutions of his-

tidyl-cysteinyl-glycine and phenyl-cysteinyl-glycine as an NMR reference,

and, for all other samples, the phenyl of the benzyl alcohol served as refer-

ence. Samples were prepared at two different hydron concentrations: there

were samples at low pH prepared by adding deuterium chloride (50 mM), in

order to be stable against de-amination [58]; and there were samples that re-

flect physiological conditions prepared with 100 mM phosphate in deuterium

chloride (DCl), in order to obtain results relevant to physiology. The shifts of

hydron dissociation constants that result from working in 2H2O were consid-

ered to be uniform for all acid base pairs, i.e. all pKa values shifted +0.4 units,

because the isotope effect is the same for all acids and bases. The pD values is

determined with a pH-Meter as pD = pH + 0.4 [59].

NMR tubes were filled within the glove box and sealed with a rubber stopper

and Parafilm. Sets of 10 identical samples were prepared and stored in a dini-

trogen-filled glove box to prevent any reaction with dioxygen. Pairs of sam-

ples were then sequentially removed from the glove box for measuring with


47

250 MHz (for GSH) or 300 MHz (all other samples) instruments from Bruker

BioSpin GmbH (Fällanden, Switzerland). 1H-NMR spectra were recorded

with a relaxation delay of 15 s. For H,H-COSY measurements, an anaerobic

glutathione solution (10 mM) in deuterium chloride (10 mM) without radical

initiator was transferred to a glove box and distributed over NMR tubes which

were sealed with stopper and Parafilm. Samples were removed from the glove

box immediately and after six days and analysed with the 300 MHz instru-

ment. Similar aerobic experiments gave comparable results despite the pres-

ence of dioxygen: The concentration of dioxygen (approx. 260 &M is much

smaller than that of glutathione (2 mM) and the concentration of products

formed from dioxygen would have been below the detection limit.

Nuclear magnetic resonance data from the free induction decay was obtained

as data points of the dimensions time and intensity, which were transformed

to frequency and amplitude. Reference compounds allowed alignment of the

chemical shifts and normalisation of the integrals of the signals investigated.

Differences between the signals directly after mixing and after several days

were plotted. Increases originate from NMR signals of fission products of

!,!'-azodiisobutyramidin-dihydrochlorid.

Chapter 2

48

2.4 Laser Flash Photolysis

Solutions of glutathione and its derivatives were freshly prepared in 100 mM

2-methyl-2-propanol solution in sodium phosphate buffer at pH 7.3. The solu-

tions, contained in gas-tight fluorescence cells were evacuated to boiling and

subsequently filled it with argon twice, then, after a further evacuation, argon

or oxidonitrogen(•) gas was added to saturation. The resulting concentration

of oxidonitrogen(•) is 1.8#10–3 M [48].

Laser flash photolysis was conducted with an Applied Photophysics LKS 50

instrument fitted with a Brilliant B Nd:YAG Laser providing a 4th harmonic

at 266 nm with 5 ns pulses. Single pulse energies were kept lower than 20 mJ

to avoid photolysis of water [60]. The photolysis of glutathione disulfide

yields two glutathione thiyl radicals [58], and that of S–nitrosoglutathione

yields a glutathione thiyl radical and oxidonitrogen(•) [61]. The reactions

were followed at 330 nm. Digital filters were used to reduce high frequency

noise.

Curve fitting was carried out with Kaleidagraph software, and kinetics con-

stants were estimated with a pseudo-first-order model, given that

[GS•] 3 [NO•].


49

2.5 Disulfide Synthesis

The synthesis has two steps: firstly, the formation of the S–nitrosothiol from

the thiol in acidic dioxidonitrate(1–) solution similar to Hart [36], and second-

ly the thermal decay of the S–nitrosothiol to the desired disulfide by heating.

Of course, thiols and S–nitrosothiols are odorous, so adequate precautions

such as a base bath are recommended. The procedure is as follows: 25 g of

high grade purity Milli-Q ice in a 50 mL separation funnel were mixed with

1 mL (yields approx. 0.5 M) concentrated hydrochloric acid and 1 mL of thiol

(approx. 0.01 mol). Just before all the ice was melted, 1 g (0.015 mol) of so-

dium dioxidonitrate(1–) was added. The separation funnel was quickly closed,

and alternatingly shaken and aerated until dioxidonitrogen(•) started emerging

upon opening the valve. The aqueous phase was poured out and the remaining

crude S–nitrosothiol of reddish colour was immediately transferred with a

Pasteur pipette to an open empty Schlenk tube attached to a slight stream of

nitrogen. The Schlenk tube was then gently heated with a “heat gun” until the

colour of the crude product had disappeared and the desired product had

formed. Residual thiol would either evaporate or react with residual dioxygen

and oxidonitrogen(•) to S–nitrosothiols.

Chapter 2

50

2.6 NMR Coalescence Measurement

Freshly prepared S–nitrosoglutathione was filled into a standard NMR sample

tube, diluted with deuterated chloroform and cooled during the transfer to the

400 MHz NMR spectrometer instrument from Bruker BioSpin GmbH

(Fällanden, Switzerland).

Individual standard 1H-NMR spectra were recorded at temperatures from

223 K to 313 K every 10 K. The energy of activation EA was calculated by

evaluating the temperature dependent curve of the peak integral ratios drawn

to an Arrhenius plot.

UV-Vis spectra were recorded on a Specord 200 instrument from Analytik

Jena (Jena, Germany) either in dilute aqueous solution or in gas phase. Gas

phase samples were a few drops of sample material placed in lockable cu-

vettes that were closed after a short evaporation time.

Chapter 3

Hydrogen Exchange Equilibria in Glutathione Radicals:

Rate Constants

This chapter has been adapted from:

Dustin Hofstetter, Thomas Nauser and Willem H. Koppenol, Chem. Res. Tox-

icol. 2010, 23(10), 1596

(Reprinted with permission. Copyright 2010, American Chemical Society)

Chapter 3

52

3.1 Introduction

Glutathione has many physiological functions. It is a substrate for the enzyme

family of glutathione S-transferases, which catalyse the formation of glutathi-

one conjugates of xenobiotics to facilitate their transport and detoxification

[62]. Stores of glutathione are also a significant source of reduction equiva-

lents, e.g., for reactions of the glutathione peroxidases to convert dioxidane to

water, with generation of glutathione disulfide [4]:

2 GSH + H2O2 GPx

GSSG + 2 H2O (3.1)

In turn, glutathione disulfide is re-reduced to glutathione by glutathione re-

ductase, with reducing equivalents from adenine dinucleotide phosphate [63]:

GSSG + NADPH + H+GSR

2 GSH + NADP+ (3.2)

GSH, which is rather abundant in the cytosol (5 – 10 mM) [3], is believed to

react to some extent with partially reduced oxygen species and partially oxi-

dised nitrogen species to form the glutathione radical. Glutathione reacts suf-

ficiently rapidly with the oxidanyl radical [27] to consider it a possible scav-

enger; rate constants for reaction of glutathione with dioxidane or diox-

ide(•1!) are much smaller, 15 M–1 s–1 [64] and 10–2 – 10–3 M–1 s–1 [65],

respectively, such that these species are not scavenged. (Dioxido)-

oxidonitrate(1%) reacts more readily with dioxidocarbon than with glutathi-

one: the products of rate constants and concentrations are approximately

39 s–1 and 1 s–1 for dioxidocarbon [28; 66] and glutathione [67], respectively.

However, dioxidonitrogen(•) and trioxidocarbonate(•1!) radicals produced in

the reaction of (dioxido)-oxidonitrate(1%) with dioxidocarbon may react with

glutathione[68; 69].

A considerable proportion of oxidanyl(•), dioxidonitrogen(•), trioxidocar-

bonate(•1!) and (dioxido)-oxidonitrate(1%) radicals react with proteins and

Hydrogen Exchange Equilibria in Glutathione Radicals: Rate Constants

53

membrane lipids to produce protein and lipid radicals. Although lipid radicals

can be repaired by vitamin E [70; 71], protein radical repair by glutathione,

though not very efficient [72], would generate additional glutathione thiyl

radicals.

Thus, several pathways lead to formation of the glutathione thiyl radical,

which is generally assumed to react under in vivo conditions with the gluta-

thione thiolate anion to form the glutathione disulfide radical anion [24; 15];

subsequent reaction of glutathione disulfide radicals with dioxygen and dis-

posal of dioxide(•1%) by superoxide dismutase terminates this radical chain

reaction [73]. The formation and reactivity of both glutathione thiyl radi-

cals and glutathione disulfide radical types have been studied for nearly a half

a century [74; 75; 22; 58]. In the 1980s, it was reported that not only S-

centred radicals but also C-centred radicals are part of the chemistry of the

glutathione thiyl radical [76]. Ten years later, it was established that the S-

centred and !C-centred radicals are in intramolecular equilibrium [17]:

HGS• 2 •GSH (3.3)

Details of this reaction have been established for cysteine, glutathione and

other oligopeptides [15; 18; 19]. It has been demonstrated that irradiated lyso-

zyme undergoes fragmentation [77], probably after migration of an electron

from an !-carbon to the initially formed Cys thiyl radical. Subsequent reac-

tions, yet to be identified, lead to the observed fragmentation [19]. In spite of

these reports, the notion that glutathione thiyl radicals are innocuous is persis-

tent.

We reduced glutathione disulfide by radiolytically generated hydrogen atoms

and hydrated electrons under acidic and basic conditions in order to determine

rate constants and equilibrium constants for the reversible intramolecular hy-

drogen exchange between S-centred and C-centred glutathione radicals. We

re-interpret published pulse radiolysis data of glutathione and show that the

Chapter 3

54

formation of C-centred radicals takes place extremely rapidly, but also that

more than one C-centred radical species participates in reaction 3.3. While

fast formation of !C-radicals from S-radicals is reported in the literature [17;

78], we propose the additional formation of "C- (or $C-) radicals. These reac-

tions have been investigated under both alkaline and acidic conditions and

will therefore also take place at neutral pH.


55

3.2 Results

In Fig. 3.1, as proof of concept, we show the quantitative formation of gluta-

thione disulfide radicals [16; 24] (" = 8#103 M–1 cm–1 at %max = 425 nm) [24]

by reaction of e–aq and H• with 50 mM glutathione disulfide at pH 9.7 in the

presence of 10 mM GSH / GS– and 1 M tert-butanol to scavenge oxidanyl(•)

quantitatively. In the presence of glutathione, reactions –3.4 and 3.5 are fa-

voured, and reaction 3.4 is suppressed [24; 79]:

GSSG•– GS– + GS• (3.4)

GS(H)SG• GSSG•– + H+ (3.5)

Figure 3.1: Spectra of an Ar-saturated 50 mM GSSG solution at pH 9.7 at 1 "s (#) and 8 "s ($) after irradiation (36 – 41 Gy) in the presence of 10 mM GS–/GSH and 1 M tert-butanol. The absorbance is normalised to a dose of 1000 Gy. No measure-ments below 300 nm are possible as the added thiolate blocks light transmission.

Chapter 3

56

At concentrations of an electron scavenger (glutathione disulfide in this case)

as large as 50 mM, the expected radiochemical yield (G-value) has to be cal-

culated on the bases of Wardman-Asmus-Schuler formula [80] and the data of

Balkas et al [81]:

With G(e-aq) = 3.6 so calculated and G(H•) = 0.6, we expect formation of

17.3 &M ([GS•] + [GSSG•–] = [S]) at a dose of 42 Gy. Given K8 = 5 # 10-4 M

(Mezyk, 1996), we expect [GSSG•–]/[GS•] = 100. The amount of glutathione

disulfide radicals found corresponds to a quantitative yield of reactions –3.4

and 3.5 according to c = & • D • G • 0.1036 'M, in which c is the concentra-

tion of primary radicals interacting with glutathione disulfide, & is the density

(1 kg l-1), D is the dose, and G is the sum of G(e–aq) and G(H•) [55]. The gluta-

thione disulfide radical is too short-lived to be observed because the equilibri-

um of reaction 3.5 lies to the right (pKa = 5.9) and is attained nearly instanta-

neously [24]. We expect an initial absorbance at 425 nm of 137 mAbs, in ex-

cellent agreement with the measured 131±4 mAbs.

All other experiments were carried out in the absence of glutathione. Because

only 1 mM glutathione disulfide was present, we used the standard value for

G(e-aq), 2.75. Spectral changes at pH 11.8 (Fig. 3.2) show the conversion of

the glutathione disulfide radicals at 420 nm to one or more species that have

large, increasing absorptivities at wavelengths lower than 300 nm. The spec-

trum at % > 430 nm at 0.75 &s appears to show a contribution from the hydrat-

ed electrons. The build up of absorption at 240 nm and at 280 nm (Fig. 3.3)

have different half-lives, 7 &s and 4 &s, respectively and the maxima are

reached at different times. This is evidence for at least two concurrent pro-

cesses. Importantly, the spectral signatures of these processes are consistent

with the formation of both !C- and "C-type radicals [82].

8s

8s

aq106/]S[k1

106/]S[k23.255.2)e(G

!+

!+="


57

Figure 3.2: Spectra of Ar-saturated 1 mM GSSG solution at pH 11.8 after irradiation (56 – 66 Gy) in the presence of 1 M tert-butanol. %, 0.75 "s; !, 4 "s; #, 15"s, and $, 25 "s. The absorbance at 0.75 "s is due to GSSG•–. The absorbance is normalised to a dose of 1 kGy.

Figure 3.3: Time-dependent changes in absorbance at &, 240 nm; !, 280 nm; #, 330 nm, and $, 420 nm of an Ar-saturated 1 mM GSSG solution at pH 11.8 after irradiation (56 – 66 Gy) in the presence of 1 M tert-butanol. The absorbance is normalised to a dose of 1 kGy.

Chapter 3

58

Figs. 3.4 and 3.5 show the results of pulse radiolysis of glutathione disulfide

at pH 2; at wavelengths <300 nm, the initially increasing absorbance at lower

wavelengths is transformed to a shoulder at ca. 260 nm (Fig. 3.4). The spectra

in Fig. 3.4 could indicate that a fast equilibrium is established between radical

species that are subject to further reaction. If so, isosbestic points could be

imagined at ca. 250 nm and 310 nm. While the kinetics traces at 260 nm (not

shown, but see Fig. 3.4) and 280 nm are similar, they are clearly different

from that at 240 nm (Fig. 3.5). The absorbance of the thiyl radicals at 330 nm

[19] seen at 0.75 &s decays to a residual absorbance, presumably of the spe-

cies that has an absorbance maximum at ca. 265 nm. At 410 nm, the absorb-

ance maximum of the glutathione disulfide radical, the decay is complete in

2 &s. Thus, under acidic conditions, we also find evidence for formation of

both !C- and "C-type radicals.

We determined rate constants both at 260 and 280 nm and found them to be

identical, but because the signal to noise ratio is superior at 260 nm, we rely

on measurements taken at that wavelength. The rate constants for product

formation at pH 2 is kobs = (1.0 ± 0.5)#106 s–1 and at pH 11.8,

kobs = 2.6 ± 0.3#105 s–1.


59

Figure 3.4: Spectra of an Ar-saturated 1 mM GSSG solution at pH 2 after irradiation (23 – 30 Gy) in the presence of 1 M tert-butanol. %, 0.75 "s; !, 4 "s; #, 15"s, and ", 25 "s. The ab-sorbance is normalised to a dose of 1000 Gy.

Figure 3.5: Time-dependent changes in absorbance at &, 240 nm; !, 280 nm; #, 330 nm, and $, 420 nm of an Ar-saturated 1 mM GSSG solution at pH 2 after irradiation (56 – 66 Gy) in the presence of 1 M tert-butanol. The absorbance is normalised to a dose of 1000 Gy.

Chapter 3

60

3.3 Discussion

The main conclusion that we can draw from the work we present here is that,

after initial formation of the glutathione thiyl radical, hydrogen transfer leads

to formation of both !C- and "C-centred radicals in alkaline and in acidic solu-

tion. Whereas in alkaline solutions formation of the !C-radical of Glu with

subsequent de-amination has been reported [18], this process is expected to be

slower in acidic solution, because the protonated ammonium group deac-

tivates the !C-hydrogen. "-Elimination from Cys !C-radical could lead to

desulfurisation [83], a process that takes place at a rate that is at least one or-

der of magnitude slower than the processes discussed here (T. Nauser, 2010,

unpublished).

The reaction of e–aq and H• with glutathione disulfide to produce glutathione

disulfide radicals is quantitative, and the subsequent generation of glutathione

thiyl radicals is straightforward. We chose this method because of literature

reports that the reaction of oxidanyl(•) radicals with glutathione produces both

S- and C-centred radicals by H•-abstraction [16; 85]. A clean formation of

glutathione thiyl radicals from glutathione disulfide radicals implies that any

other species observed must be a product of glutathione thiyl radicals. Thus,

any C-centred radical on glutathione originates from intramolecular hydrogen

transfer to a glutathione thiyl radical. We observed no long-lived (t1/2>10–4 s)

adduct at 380 nm, which excludes any contribution of the bimolecular reac-

tion of glutathione thiyl radicals with glutathione disulfide [86].

In addition to the equilibrium between S-centred and C-centred radicals in

glutathione (reaction 2.3), the glutathione C-centred radical is also subject to

acid-base equilibrium [17]:

•GSH •GS% + H+ (3.6)


61

The rates of establishing all equilibria between carbon- and sulfur-centred

radicals, is represented by the observed rate constant kobs i.e., reaction 3.3, is

the sum of the forward and reverse rate constants [87]. Notably, at pH 11, the

availability of glutathione C-centred radicals is dependent on reaction 3.6,

thus k–3.3 is multiplied with K3.6. Given that the absorptivities of •GSH and •GS– at 260 nm are similar—the absorbing chromophore remains un-

changed—kobs for formation of (•GSH + •GS–) may be written:

kobs = k3.3 + k–3.3 • K3.6–1 (3.7)

The protonation equilibrium of the glutathione radical, pK3.6 is expected to be

very similar to pKa (GSH) 9.2 [5] thus K3.6 = 400 at pH 11.8: As an electron

deficient compound, the pKa of glutathione C-centred radicals could be even

lower, which would strengthen our argument that kobs = k3.6 at pH 11.8. Since,

at pH 2, the thiol is fully protonated, kobs (pH 2) = k3.3 + k–3.3. The difference

between the observed rate constants at pH 2 and pH 11.8 is, thus:

/kobs = kobs (pH 2) – kobs (pH 11.8)

= k–3.3 · (1 – K3.6–1) , k–3.3 = 1#106 s–1 – 2.6#105 s–1 = 7#105 s–1.

In this way, we calculate that k3.3 = kobs (pH 2) – 7#105 s–1 = 3#105 s–1, and

K3.3 , 0.4. Thus, at equilibrium, the fraction of C-centred radicals is approxi-

mately 30%.

These rate constants are of the same order of magnitude as those determined

for N-Ac-Cys-Gly6 and similar molecules [19]. From the absorbance versus

time curves in Figs. 3.3 and 3.5, it can be seen that a strong absorption is al-

ready present within microseconds after the pulse. Since glutathione thiyl rad-

icals absorb weekly in that region [17] and glutathione disulfide radicals

would exhibit a maximum at 425 nm, we must assign this absorption to the

presence of the C-centred radicals in substantial amounts. In proteins, such

hydrogen abstractions could, thus, lead to irreversible damage via formation

Chapter 3

62

of carbon-carbon bonds or backbone fragmentation, as has been shown for

lysozyme [77].

Closer inspection of the spectra in Figs. 3.2 and 3.4 reveals that shapes of the

absorption bands change with time, with higher absorbance towards lower

wavelengths initially and later a shoulder at approximately 260 nm. Also, the

maximum absorbance of the traces at 240 nm and 280 nm is reached at a dif-

ferent time. According to Neta et al. [82], the spectral characteristics of !C-

and "C-type radicals are different, with !C-radicals exhibiting maximal ab-

sorbance at ca. 265 nm and "C-radicals having increased absorbance at lower

wavelengths. The UV-Vis patterns observed allow us to attribute only the

type of radical, i.e. !C- or "C- radicals, but not to precisely identify the residue

at which the C-centred radical is located: The lack of reference spectra does

not allow us to exclude the formation of $C- radicals.

At pH 2 (Fig. 3.4), the first recorded spectrum already shows the signature of

a "C-radical. Within microseconds, an absorbance with a shoulder at ca.

260 nm appears, which we ascribe to the !C-radical. A similar pattern is also

found at pH 11: here, too, the "C-radical is formed initially, but immediate

deprotonation (reaction 3.6) of the thiol inhibits reaction –3.3 and confines the

radical: no spectral signature of the !C-radical is observed.

The comparison of the spectra confirms K3.3 , 0.4: at pH 2 (Fig. 3.4) the C-

centred radicals expose a normalised absorbance at 260 nm of 0.37 mAbs,

which is approximately 30% of the 1.1 mAbs normalised absorbance at the

same wavelength at pH 11.8 (Fig. 3.2), where the vast majority of the radicals

are confined to their "C-form.

Abedinzadeh et al. [15] made similar observations upon generation of gluta-

thione thiyl radicals from glutathione and oxidanyl(•) radicals or Br2•–. They

determined two different rate constants for product build-up at 270 nm and

320 nm, and explained their findings with a reaction scheme in which the oxi-


63

dant attacks glutathione at different sites with different rates, with direct pro-

duction of both S- and C-centred radicals. However, according to such a reac-

tion scheme, mathematics requires that all products must be formed with the

same rate constant, namely ksum, which is the sum of all rate constants in-

volved [88]. Intuitively, this can be rationalised, because the kinetics are de-

termined by the component present in “subcess” i.e. oxidanyl(•) radicals or

Br2•!. This requirement is not met, both by their and our experimental data,

The sequential evolution of absorptions observed by Abedinzadeh et al. is

better described with a sequential evolution of products, e.g. initial formation

of an S-centred radical ("max = 330 nm) that reacts to form, and is in equilibri-

um with, a C-centred radical ("max , 265 nm).

Our data also indicate that there is interconversion between !C- and "C-

radicals (Fig. 3.4). The "C-centred radical is kinetically favoured, and the !C-

centred radical is the thermodynamically preferred C-centred radical: experi-

ments at pH 11.8 show that only the presence of "C-radicals (Fig. 3.2); since

both C-centred radicals are formed at pH 2, we assume that the interconver-

sion of the C-centred radicals at lower pH proceeds via the S-centred radical:

!G•–SH HG–S• "G•–SH (3.8)

Such reactions are known in organic chemistry and are described as polarity

reversal catalysis [89; 90]. Using published values for spectra of C-centred

radicals derived from #-chloroalanine [82] (Fig. 3.2), we can make a rough

estimate of the ratio of !C- and "C-centred radicals: the spectra of the experi-

ments obtained at pH 2 at 20 &s are most suitable, because, at that pH, the

spectral signatures of both radical types are rapidly formed, and, after 20 &s,

no further increase in absorbance is observed. Thus, H-exchange equilibria

are rapidly established. With 1650 M–1 cm–1 and 730 M–1 cm–1 for the molar

absorptivities of "C-centred radical at 240 nm and 260 nm ([82], Fig. 3.2), re-

Chapter 3

64

spectively, and with 1400 M–1 cm–1 and 2000 M–1 cm–1 for those of the !C-

centred radical at 240 nm and 260 nm ([82], Fig. 2.2), respectively, we obtain

a value of ca. 3:1 for the ratio of "C/!C-radicals directly after the decay of the

glutathione disulfide radicals. With this we achieve the first quantification of "C-radicals in peptides, although ab inito calculations at B3LYP/6-31G(d)

level predicted their existence as unlikely [91]: this otherwise very carefully

executed study neglected to calculate transition state energies of thermody-

namically less favoured products, which probably is the case with "C-radicals.

In the literature, one finds, that ab inito calculations with more elaborate

computational basis sets show deficiencies in accurate predictions of RSNO

structures [38], which can be another reason for the mismatch of calculation

and experimental results.

Our findings show that, after initial formation of the glutathione thiyl radical,

S-centred, !C-centred, and "C-centred radicals undergo equilibrium intercon-

version. Thus, oxidation of Cys residues in proteins could produce C-centred

radicals that undergo reactions other than disulfide bridge formation, ultimate-

ly leading to fragmentation or polymerization. Such processes have been ob-

served after disulfide $-radiolysis in insulin [92; 77] “repair” of a C-centred

radical via H-atom transfer, intramolecularly or from an antioxidant such as

ascorbate, is expected to result in partial racemization at that carbon, with po-

tentially serious consequences [93].

The relative stability of !C-radicals was predicted [78; 94; 17] and demon-

strated [95] earlier. Here we show that, in addition, "C-radicals are formed

and that they are surprisingly stable. At present we try to locate the various

types of radicals on glutathione.


65

3.4 Acknowledgment

We would like to thank Patricia L. Bounds for her extensive help with lan-

guage editing on this article. This work was supported by ETH Zürich and the

Swiss National Science Foundation.

Chapter 4

Hydrogen Exchange Equilibria in Thiols — Sites

This chapter is in preparation for publication as:

Dustin Hofstetter, Basil Thalmann, Thomas Nauser, and Willem H. Koppenol,

Chemical Research in Toxicology

Chapter 4

68

4.1 Introduction

The formation of peptide and protein thiyl radicals has attracted attention over

the last decades [74; 75; 22; 17; 15; 58; 93; 19]. This is mostly because thiols

in general, and glutathione in particular, are very important compounds in

human metabolism: glutathione is involved in detoxification and it provides

reducing equivalents in order to maintain the redox state of the cell. Further-

more it seems to have an important role in ageing, particularly in interaction

with partially reduced oxygen species or partially oxidised nitrogen species.

Although (dioxido)-oxidonitrate(1%) and dioxide(•1%) can be excluded as

generators of glutathione thiyl radicals—their reactions are too slow [64; 96;

28], or they react with other cellular components—glutathione thiyl radicals

can be produced from of the reaction of glutathione and oxidanyl(•) radicals

[27], dioxidonitrogen(•) [28], trioxidocarbonate(•1%) [97] or membrane lipid

radicals [30].

Recent pulse radiolysis experiments showed [98] that our understanding of

the reactivity of the glutathione and cysteine thiyl radicals is incomplete. Alt-

hough de-amination [58] and desulfurization [83] are probable fates of such a

radical: there seems to be a plurality of co-existing intermediate radical spe-

cies centred at sulfur, !-carbons and "-carbons [98]. In the case of glutathi-

one, kinetic methods do not tell us on which amino acid an !- or "-radical is

located.

In order to determine these sites, we carried out 1H-NMR experiments analo-

gous to those conducted with cysteamine [26] to determine which hydrogen

atoms are abstracted and to what extent. Briefly, radicals generated from de

thermal decomposition of an azo-compound abstract a hydrogen atom from

the thiol, reactions 1 and 2. Alcohols can act as a radical chain extender, reac-

tion 4.3:


69

R–N=N–R * 2 R• + N2 (4.1)

R• + R2SH ! R2S• + RH (4.2)

R2SH + •R1OH ! R2S• + HR1OH (4.3)

At optimum concentration ratios of radical chain initiator !,!'-

azodiisobutyramidin-dihydrochlorid (AAPH), chain extender (alcohol) and

target molecule (thiol), the radical chain will be long and R2S• can be generat-

ed many times:

The radical chain is initiated by the thermal decay of !,!'-

azodiisobutyramidin-dihydrochlorid and immediately promoted to the thiols,

reactions 4.4 and 4.5:

R–N=N–R * 2 R• + N2 (4.4)

R• + R2SH ! R2S• + RH (4.5)

The radicals are produced in rates of nmol s–1 [99], so sufficient thiyl radical

generation can only be achieved by chain prolongation e.g. with an alcohol,

which can be viewed as a radical carrier link between two thiyl radicals, equi-

librium 4.6:

R2SH + •R1OH ! R2S• + HR1OH (4.6)

The concentration of all reactants had to be optimised: at high concentrations

of chain carrier (i.e. alcohol) equilibrium 4.6 can shifted too far to the right

and either the concentration of thiyl radicals is too low to form enough prod-

ucts or the lifetime of thiyl radicals is too low and inhibits intramolecular hy-

drogen transfer. On the other hand, if the alcohol concentration is too low, the

chain length is too short for sufficient product build-up. Only optimised con-

ditions allow to delay the termination reactions 4.7 – 4.9 and to achieve a

measurable product concentration.

2 R• * R–R (4.7)

Chapter 4

70

R• + R’S• * R’S–R1 (4.8)

[R’SSR’]•- + R• + H2O * R’SSR’ + RH + HO– (4.9)

Reactions 4.7 and 4.8 effectively eliminate thiol from the reaction cascade,

additionally lowering yields.

Usually acidic systems with pD < 3 are easiest to handle because equilibrium

4.10 is shifted far to the reactant side:

R’S• + R’S– ! [R’SSR]•– (4.10)

Experiments at higher pD often are very tricky. It should be noted here, that

all shown experiments can be used for positive proof only: Because of the

complex nature of the underlying chemistry one can never be sure not to have

missed the required reaction conditions if no products are found. In the pub-

lished literature [26], competing intermolecular reactions were studied. Here

we face competing inter- and intramolecular reactions, a system which is

much more challenging.

These experiments are carried out in deuterated water. From NMR experi-

ments the decrease of H (1H) signal intensity is used to deduce where H has

been replaced by the NMR-invisible D (2H) from the solvent via protolysis of

the thiol group, reactions 4.11 and 4.12:

HR’S• ! •R’SH (4.11)

•R’SH + D2O ! •R’SD + HDO (4.12)

•R’SD ! DR’S• (–4.11)

Experiments were carried out in acidic and neutral media. The results confirm

that, contrary to widespread opinion, thiyl radicals are not harmless.

In addition, we find evidence that glutathione re-arranges in acidic media.

Although C–H connectivities and signal integrals are retained, the signal posi-

tions shift significantly in the course of time.


71

4.2 Results

The glutathione thiyl radical at neutral pH abstracts the hydrogen at the Cys !

site‡. During 96 hours, the NMR integral of the !-H decreases to ca. 50%

(Fig. 4.1). No other exchanges are observed (Fig. 4.2). Surprisingly, at acidic

pH, the "- and $-sites of Glu exchange H• with the thiyl radical, but no other

reactions can be seen (Fig. 4.2). No H•-exchange is observed in cysteine

(Fig. 4.2), the changes are, both at pH 1 and pH 7, not significant. However,

when a glycine residue is present at the C-terminus, the Cys "-carbon of cys-

teinyl-glycine shows significant H/D-exchange at neutral pH, while no signif-

icant changes are observed at other sites. No exchange activity is observed in

cysteinyl-glycine at low pH (Fig. 4.2).

Figure 4.1: NMR Signal (black line) and integral (gray line) for the hydrogen at the glutathione (2 mM) Cys ' site at pH 7 im-mediately after preparation (left) and after 96 h (right) at 24°C.

‡ The Greek letters refer to the position of the C-atom from the amine group in the amino acid residue. IUPAC numbering is in roman letters starting at the C-terminus of the amino acid chain, different amino acids in a peptide chain are distinguished by an increasing number of primes: The 2-site according to IUPAC is equal to Cys ! in Cys, to Glu ! in GSH, and to Gly ! in Cys-Gly, Phe-Cys-Gly and His-Cys-Gly. The 3-site is equal to Cys " in Cys, to Glu " in GSH. The 2’ and 3’-sites are equal to Cys ! and Cys " in GSH, Cys-Gly, Phe-Cys-Gly and His-Cys-Gly.

Chapter 4

72

Figure 4.2: NMR Signal change per H atom at the glutathione sites (with 2( error bars) during radical exposure. Cys and Cys-Gly at pH 1 (20 d) and 7 (15 d). Glutathione at pH 2 (192 h) and pH 7 (96 h), due to signal overlap, the glycine and gluta-mine ' sites, and glutamine ) and * sites could not be separat-ed in both spectra. His-Cys-Gly at pH 1 (53 d), H1 designates the H atom linked to the carbon atom between the nitrogen at-oms. Phe-Cys-Gly at pH 1 (53 d), The NMR signals for H atoms at meta and para positions could not be resolved. Sites with significant exchange are marked with their letter of position in the structures given.


73

Exchange also occurs in histidyl-cysteinyl-glycine and phenylalanyl-

cysteinyl-glycine, but the exchange patterns are different (Fig. 4.2): with the

exception of the His and Phe ! sites (which would correspond to the $-site of

Glu in glutathione) nearly all carbon atoms appear to exchange. In both

tripeptides, exchanges involving the Gly !- and Cys "-hydrogen are the most

significant along with those at His-H1 for histidyl-cysteinyl-glycine and o-

Phe for phenylalanyl-cysteinyl-glycine. For the Cys ! hydrogen of histidyl-

cysteinyl-glycine the results were too inaccurate to allow a conclusion. Most

important is the degree of exchange: although histidyl-cysteinyl-glycine and

phenylalanyl-cysteinyl-glycine are very similar, e.g. they both contain an ar-

omatic system, histidyl-cysteinyl-glycine exchanges are approximately twice

as large as those of the phenylalanyl-tripeptide.

As an unexpected result, new peaks were found in the 1H-NMR experiment

with glutathione after 6 d in acidic solution with or without radical initiators

being present. H,H-COSY experiments revealed that in this process the C-H-

framework of glutathione remains unchanged. Moreover, it allowed assign-

ment of the product peaks to the original peaks (Fig. 4.3) which enabled us to

correct the original peaks for shifts caused by the re-arrangement. All cross

peaks of the 2D-NMR diagram were assigned and therefore the formation of

other products can be excluded. Thus, we only observe replacement of H by

D and a slow rearrangement of glutathione.

The H• exchange is faster than the re-arrangement and therefore we believe

the results for acidic solutions of glutathione to be valid, but to be of a quali-

tative nature.

Chapter 4

74

Figu

re 4

.3:

2D H

-NM

R o

f glu

tath

ione

in D

2O in

0.1

M D

Cl a

fter

6 d

with

out r

adic

al in

itiat

or. G

luta

thio

ne u

nder

-go

es a

n un

iden

tifie

d re

-arr

ange

men

t in

acid

ic m

edia

. NM

R re

sults

sho

w th

at th

e co

nnec

tivity

of C

and

H a

tom

s re

mai

ns.

All

thre

e re

sidu

es o

f gl

utat

hion

e ar

e di

stin

guis

habl

e by

the

ir co

uplin

g sc

hem

e: G

ln p

osse

sses

thr

ee

coup

ling

prot

ons,

Cys

onl

y tw

o an

d th

e pr

oton

of G

ln d

oes

not c

oupl

e to

any

oth

er p

roto

n. 2

D H

-NM

R a

llow

s to

at

tribu

te th

e nu

mbe

r of

cou

plin

g pr

oton

s an

d th

eref

ore,

em

ergi

ng s

igna

ls c

an b

e co

nnec

ted

to th

e tra

nsfo

rmed

re

sidu

es G

ln',

Cys

' and

Gly

', re

spec

tivel

y. T

he c

oupl

ings

are

repr

esen

ted

with

the

follo

win

g lin

e ty

pes:

Cys

: wid

e da

sh, C

ys':

dash

-dot

, Gln

: sol

id, G

ln':

fine

dash

. Gly

and

Gly

': no

ne (

no c

oupl

ing)

. Cor

resp

ondi

ng s

igna

ls w

ere

adde

d fo

r eva

luat

ion

of th

e H

/D e

xcha

nge.


75

4.3 Discussion

The general concept of exchanging H-atoms with D-atoms has been illustrat-

ed earlier [26; 95; 93]. The decreasing integral in Fig. 4.1 shows the confirma-

tion of this concept in glutathione. Noise and relaxation effects caused the

signals to be of inhomogeneous quality. We therefore present our results with

an error of two standard deviations, to prevent misinterpretation. Neverthe-

less, our experiments show that H/D exchange takes place in all peptides in-

vestigated at neutral pH and, for glutathione, histidyl-cysteinyl-glycine and

phenylalanyl-cysteinyl-glycine, also at acidic pH. This supports our previous

finding [98], where we determined intramolecular H-abstraction rates and

equilibria involving thiyl radicals, but could not locate the H-atoms concerned

and the present work assigns them to a specific site in the molecule. The dif-

ferent distribution patterns between !- and "-centred carbon radicals of this

and the previous finding are note contradictive: the amount of incorporation

of deuterium atoms into glutathione is dependent neither on persistence, con-

centration or fast equilibration of carbon-centred radicals of glutathione but

on the turnover, of which the rate constant is equal to k!/4/k" · (k! + k")–1.

The visibility of the carbon centred radicals in the UV-spectrum depends on

both the equilibrium constant K = k!/k" and the molar absorptivity, the equi-

libration rate is equal to the sum of the rate constants k! + k", and the persis-

tence of a carbon-centred radical increases with decreasing k". Therefore nei-

ther a high equilibration rate constant nor a high persistence of carbon-centred

radicals necessarily leads to a high turnover rate constant. Efficient H/D ex-

change, the basis of a significant change in the NMR signal, needs both high

k! and high k". As intramolecular H-abstraction in thiyl radicals is not lim-

ited to glutathione these observations are of major interest.

Our experiments showed where H-abstraction takes place in vitro, and

could occur in vivo, but they do not show a discernible pattern. Proximity is

Chapter 4

76

not an important factor because Cys " is abstracted in cysteinyl-glycine, and

Cys ! is abstracted in glutathione (and possibly in Cys), at pH 7. It is clear

that: abstraction is dependent on the pH-value, i.e. the abstraction patterns are

different under acidic and neutral conditions. Whereas the at pH 7 the abstrac-

tion of the hydrogen atom the Cys ! site can be explained by proximity and

the formation of a (-system, the abstraction pattern at pH 2 is very surprising:

abstraction of hydrogen atoms at Glu " or $ position does not seem very intui-

tive and lacks any obvious driving force. However, the experiments with his-

tidyl-cysteinyl-glycine and phenylalanyl-cysteinyl-glycine show abstractions

at equivalent positions. Second, we also observe that compounds that contain

nitrogen atoms (or, even more general, hetero atoms with lone pairs) ex-

change more than others. The comparison of histidyl-cysteinyl-glycine and

phenylalanyl-cysteinyl-glycine shows that there is more H/D-exchange in his-

tidyl-cysteinyl-glycine than in phenylalanyl-cysteinyl-glycine, although both

structures are very similar: the cysteinyl-glycine part is the same and the first

residue contains an aromatic system. However, we observe more exchange,

especially near the N-terminus of histidyl-cysteinyl-glycine. This suggests

that the availability of nitrogen or lone pairs enhances H/D-exchange by low-

ering the activation energy for H-abstraction.

RS• + R’CH ! RSH + R’C• (4.13)

It was previously assumed that sulfur radicals were relatively stable. We have

shown that such radicals produce intramolecularly, and intermolecularly [100;

93; 19] C-centred radicals. These radicals react further, as mentioned, to irre-

versibly form stable de-aminated [58] and desulfurised [83] products. Thus,

the concentration of thiyl radicals decreases rapidly with time.

GSNO may be formed by nitrosation of glutathione [37], but it is not formed

from the reaction of GS% with oxidonitrogen(•) because it yields a GSNO%

radical ion, which subsequently results in the slow formation of glutathione


77

disulfide and diazenediol (HON=NOH) [101]. It is surprising, that the in vivo

mechanism of nitrosation has not yet been elucidated.

Additional chemical implications of our findings are that peptides can irre-

versibly form intra- or intermolecular cross-links. This would increase protein

turnover, and therefore increase stress to the cell. C-centred radicals could

also serve as initiators for peroxidation of proteins. Thus, S-centred radicals

are not harmless

Although both the glutathione thiyl radical and the dioxide(•1%) radical are

formed “accidently” in vivo, there is no enzymatic pathway to deal with gluta-

thione thiyl radical, while the disposal of dioxide(•1%) radicals by superoxide

dismutases is very efficient. Glutathione thiyl radicals are rapidly reduced by

monohydrogen ascorbate [102] and urate [103].

Previously sulfur radials were thought to disappear via reaction with thiols to

yield the according disulfide radical anions, which, in turn, react with dioxy-

gen to form dioxide(•1%) radicals [73]. We already showed that carbon-

centred radicals are formed, and here which sites are to be considered vulner-

able.

The chapter of thiyl radicals in general and of the glutathione thiyl radical in

particular does not appear to be finished yet. Many processes such as H-

abstraction, de-amination [58] or desulfurization [83] determine the fate of

thiyl radicals. In order to capture important stages of processes in diseases and

in ageing, these underlying processes must be understood, and formation of "-

and $-radicals is a first step.

As for the acidic re-arrangement of glutathione, there are a variety of possible

structures. The acid catalysed formation of a thiolactone (Fig. 4.4) is in

agreement with the NMR data and the experimental conditions, but other

structures may also fulfil these requirements.

Chapter 4

78

Figure 4.4: We suggest this thiolactone as the product of the unknown re-arrangement under acidic conditions.


79

4.4 Acknowledgment

We thank Dr. Heinz Rüegger (†2010) for his engaged and extensive help with

NMR technique and instruments. This work was supported by ETH Zürich

and Swiss National Science Foundation.

Chapter 5

The Glutathione Thiyl Radical Does not React

with Nitrogen Monoxide

This chapter has been published as:

Dustin Hofstetter, Thomas Nauser and Willem H. Koppenol, Biochemical and

Biophysical Research Communications 2007, 360(1), 146

(Reprinted with permission. Copyright 2007, Elsevier)

Chapter 5

82

5.1 Introduction

Nitration and nitrosation of bioactive molecules has gained importance ever

since the biological role of oxidonitrogen(•) was discovered in 1987 [39;

104]. It has become associated with oxidative stress and disease [105], subse-

quently, S-nitrosated amino acids, peptides and, proteins including cysteine,

glutathione, or serum albumin have been detected, and their biosyntheses was

tentatively attributed to reaction with oxidonitrogen(•) [106]. The crucial step

in S–nitrosothiol biosynthesis is believed to be the reaction of oxidonitro-

gen(•) with a thiyl radical [46; 107]:

RS• + NO• % RSNO (5.1)

To date, only an estimate exists for the rate constant for Eq. (5.1), namely

k5.1est & 109 M–1 s–1 [46; 108], and this value has been used in various models

[46; 107; 108].

One particularly abundant thiol in the cell is glutathione [109]. Its S-nitrosated

derivative is also relatively abundant in vivo and does not decompose in

aqueous solution at room temperature [50; 35]. Because of its facile synthesis,

S–nitrosoglutathione has been widely studied.

The concentration of oxidonitrogen(•) in the physiological environment is ap-

proximately 10–7 M [40], which, combined with k5.1est, gives a first-order rate

constant of k’5.1est & 102 s–1. However, a competing reaction, namely the intra-

molecular radical rearrangement [110] must also be considered:

GS• # •GS (5.2)

In this hydroxide-dependent re-arrangement, the thiyl radical abstracts a H•

atom from an accessible tertiary carbon proximal to a carboxylate group. The

equilibrium of Eq. (5.2) lies to the right, and the rate constant k5.2 is approxi-

mately 103 s–1 at physiological pH [17]. A comparison of k’5.1est and k5.2 shows

The glutathione thiyl radical does not react with nitrogen monoxide

83

that a glutathione thiyl radical is more likely to rearrange to glutathione car-

bon-centred radical than to react with oxidonitrogen(•).

In this work, we derive an upper limit of (2.8 ± 0.6)#107 M–1 s–1 for k5.1, much

lower than the estimate of 109 M–1 s–1 and much lower than expected for a

radical reaction.

Chapter 5

84

5.2 Results

After flashing with a laser pulse, the absorption of S–nitrosoglutathione

(!330 nm = 850 cm–1 M–1) in an argon-saturated solution bleaches immediately

(Fig. 5.1), but this absorption does not recover to the original level within

10–4 s. This shows that S–nitrosoglutathione undergoes homolysis to form ox-

idonitrogen(•) and the glutathione thiyl radical (!330 nm = 580 cm–1 M–1 [24]),

but does not regenerate. The same experiment carried out with oxidonitro-

gen(•) instead of argon shows the same outcome. Even under conditions of

excess oxidonitrogen(•) (1.8#10–3 M), the formation of S–nitrosoglutathione

is not observed.

Figure 5.1: Laser flash photolysis observed at 330 nm of 1.2+10–3 M S–nitrosoglutathione in argon (A) and oxidonitro-gen(•) (B) saturated solutions. Both experiments show a bleaching after the laser pulse without relaxation within 10–4 s. Even at a oxidonitrogen(•) concentration of 1.8 mM, the gluta-thione thiyl radical and oxidonitrogen(•) radicals do not appear to recombine.


85

When 2#10–3 M glutathione disulfide (!330 nm < 1 cm–1 M–1) is irradiated in

argon-saturated solution, the immediate formation and decay of the glutathi-

one thiyl radical (Fig. 5.2, A) is observed. The subsequent increase in absorp-

tion can be assigned to formation of a carbon-centred radical species [17]. In

the presence of oxidonitrogen(•), the observed absorption change is larger. At

the absorption maximum of S–nitrosoglutathione, 330 nm, the difference

spectrum (Fig. 5.2, B) reveals that a second process occurs in the oxidonitro-

gen(•) saturated solution. If we assume that this difference is caused by reac-

tion (5.1), we obtain k5.1 0 2.8 ± 0.6#107 M–1 s–1.

Figure 5.2: Laser flash photolysis observed at 330 nm of 2+10–3 M glutathione disulfide in argon and oxidonitrogen(•) saturated solutions, respectively (A). Both experiments show an initial decay and a subsequent build-up. The difference be-tween the traces (B) shows that there is an additional formation process that takes place under oxidonitrogen(•) only.

Chapter 5

86

5.3 Discussion

The results of our study do not allow us to speculate about the biosynthesis

pathway of S–nitrosoglutathione. However, our results do rule out a direct

radical mechanism. The derived upper limit of 2.8 ± 0.6#107 M–1 s–1 for k5.1 is

much lower than expected for a radical mechanism, and the first-order rate

constant in the presence of a physiological oxidonitrogen(•) concentration

would be 2.8 ± 0.6 s–1. The rate of equation 5.2 is about 300 times faster than

that of equation 5.1, even in the presence of 1.8#10–3 M oxidonitrogen(•),

and, for that reason, equation 5.1 does not likely take place under physiologi-

cal conditions. This conclusion, however, holds for homogeneous solution

only; one could imagine that equation 5.1 could proceed within the active site

of an enzyme. Given that the number of tertiary carbons in a protein that carry

an '-carboxylate group, is large, a glutathione thiyl radical is more likely to

abstract a hydrogen from protein than to react with oxidonitrogen(•) at said

active site. Biological S–nitrosoglutathione formation must, therefore, proceed

by a route other than a radical-radical reaction.

We conclude that no S–nitrosoglutathione is formed from oxidonitrogen(•)

and glutathione thiyl radicals because the interconversion of glutathione S-

centred radicals to C-centred radicals [58; 95] is very rapid and because the

reaction of oxidonitrogen(•) with glutathione thiyl radicals is unexpectedly

slow.

Given the rapid redistribution of the unpaired electron in the glutathione thiyl

radical (Chapter 4) our observation may now make sense: reaction at the C-

centred radicals which produces C–NO bonds pull the equilibrium further to

the C-centred side, which inhibits the recombination of glutathione thiyl radi-

cals and oxidonitrogen(•). However, it was shown by $-radiolysis and compe-

tition kinetics that oxidonitrogen(•) disappeared at a rate that was commensu-

rate with a rapid recombination of glutathione thiyl radicals and oxidonitro-


87

gen(•), and a S–nitrosoglutathione-like build-up was directly measured when

glutathione thiyl radicals were produced by pulse radiolysis in oxidonitro-

gen(•)-containing solution [111]. Although the discussion accepted the for-

mation of C-centred radicals and their sufficiently fast reaction with oxido-

nitrogen(•) [112], these investigations did not include products from that side

reaction: the competitively measured rate constant may still be accurate for

the formation of both, S–NO and C–NO bonds. Although the mass balance

for the reactant oxidonitrogen(•) holds in these experiments, it is not the same

for the product S-nitrosoglutathione, because analysis showed that it was

formed, but not that it was the only product. It also seems noteworthy that the

rate constants for the reaction of oxidonitrogen(•) with glutathione thiyl radi-

cals (3#109 M-1 s-1) and with C-centred radicals (3#109 M-1 s-1) [112] are

within the error.

Moreover, the m/z for all possible nitrosation products produced from the re-

distribution would be the same, rendering them inseparable by electrospray

mass spectroscopy, if indeed those unknown products would actually separate

under the chromatography conditions chosen. Thus, the matter stays unre-

solved. Most intriguingly the fate of thiyl radicals seems to be dependent on

the way they were generated: via oxidation from thiols, they seem to form

disulfides exclusively, but photolysis of disulfides yields hemi-thioacetals

[92] and one-electron reduction of disulfides leads to considerable amounts of

carbon-centred radicals [98], probable precursors of such thiohemiacetals. It

also remains unresolved, if the products found for the reaction of “thiyl radi-

cals” with oxidonitrogen(•) likewise dependent on the chemical system cho-

sen for thiyl radical production?

Chapter 5

88

5.4 Acknowledgment

This work was supported by ETH Zurich and the Swiss National Science

Foundation

Chapter 6

Synthesis of Disulfides from S–nitrosothiols

Chapter 6

90

6.1 Synthesis Letter

By the mere existence of disulfide bridges in many proteins, disulfides have

become an interesting object in biochemical related fields. The formation or

fission of such a disulfide regulates processes and the disulfide bond itself is

investigated towards its redox behaviour. To isolate the disulfide function, the

synthesis of simple derivatives can be necessary. Various methods of disul-

fide syntheses from thiols are known and established. One method involves

working in dimethyl sulfoxide with aluminium oxide as a catalyst [113], an-

other takes iodine in ethanol [114], and a third one has methanol as a solvent,

copper tetraoxidosulfate as a second reactant and sodium tetrahydridoborate

as a catalyst [115].

The formation of S–nitrosothiols via various routes is well known in general

chemistry [36]. With the discovery of the physiological occurrence of S–

nitrosothiols, their thermally induced decay to the corresponding disulfide and

oxidonitrogen(•) has also been studied [35]. Nevertheless, the use as a syn-

thetic route has never been established before. We like to communicate an

experimental manifestation of this route because it is fast, cheap and yields

high purity products without work up and despite its unconventional execu-

tion and low yields with light reactants. The procedure can be found in chap-

ter 2.6.

Generally, the disulfides do not absorb in the visible region but seemingly

show thermochromism, so that the hot product can have a slight yellow or

green absorption, whereas the cold product is colourless. The products are

sensitive to oxidation and were kept in the Schlenk tubes, they were prepared

in, and subsequently analysed by H-NMR. The measurements were in good

agreement with the literature [116; 117; 118] available. An overview is given

in Table 6.1. Overall, the synthesis can be achieved under one hour and takes

Synthesis of disulfides from S–nitrosothiols

91

only reactants of moderate or low price. All the same, it does provide poten-

tial to improve yields.

Reactant RSH Product RSSR Purity % Yield % 1 1H-NMR (CDCl3)

dodecanethiole didodecyl disulfide 77 81 0.880 (t., 6H, J = 6.38 Hz), 1.18–1.42 (m., 10H), 1.67 (quint., 2H, J = 7.25 Hz), 2.68 (t., 2H, J = 7.25 Hz)

butanethiole dibutyl disulfide 93 19 0.97 (t., 6H, J = 7.25 Hz), 1.42 (hex., 4H, J = 7.35 Hz), 1.66 (quint., 4H, J = 7.31 Hz), 2.69 (t., 4H, J = 7.38 Hz)

1,1-dimethyl-ethanthiole

(tert-butyl thiole)

bis(1,1-dimethyl-ethyl)-disulfide

97 13 1.31 (s., 18H)

2,2-dimethyl-propanethiole

(tert-pentyl thiole)

bis(2,2-dimethyl-propyl)-disulfide

96 13 0.99 (s., 18H), 2.77 (s., 4H)

Chapter 6

92

6.2 Acknowledgement

This work was supported by ETH Zurich and the Swiss National Science

Foundation

Chapter 7

Structural Properties of S–nitrosothiols

Chapter 7

94

7.1 Introduction

Ever since S–nitrosothiols were found to occur in the human body, there have

been hypotheses and speculations about the physiological role of this peculiar

type of molecule [39; 104]. The –NO group attached to the sulfur atom of a

cysteine residue suggests the involvement of oxidonitrogen(•) [106] or partial-

ly oxidised nitrogen species, which in turn caused an association of the S–

nitrosothiols with oxidative stress and disease [105].

Of major interest are the most common S–nitroso compounds of the human

physiology, mostly the derivatives of proteins containing cysteine. The most

abundant S–nitroso compound is a “simple” tripeptide S–nitrosoglutathione,

the derivative of the most abundant thiol in human cells: glutathione. Gluta-

thione consists of $-bound glutamic acid, cysteine and glycine (from N to C

terminus) and occurs up to concentrations of 10%3 mM [3]. It represents the

long-term reduction equivalent stock that in combination with various en-

zymes reduces a wide variety of compounds (cf. chapter 2). At the same time

glutathione is reduced to its disulfide.

Structural investigations on S–nitrosothiols are in a minority. This is due to

the fact that only tertiary S–nitrosothiols are stable, i.e. compounds with the

sulfur atom being bound to a quaternary carbon atom. These compounds ab-

sorb visible light at longer wavelengths and therefore are green in colour. A

typical representative of this kind is S–nitrosoacetyl-penicillamine (Fig. 7.1).

Figure 7.1: The Structure of (S)-penicillamine


95

Investigations showed that this type of compound has two main conformers,

syn and anti (Fig. 7.2) that are separated by an energy of activation of the or-

der of 1 kJ mol%1 [109]. The investigations mentioned above show, that the

anti conformer is favoured and thus the expected ratio of syn to anti conform-

ers is approximately 1:5.

Figure 7.2: View of the anti (preferred by carbons with tertiary carbon atoms) and syn conformers (preferred by compounds with less substituted carbon atoms) of the C-C-S-N dihedron. Hydrogen atoms are omitted.

Similar compounds in which the carbon atom next to the sulfur atom is less

substituted are reddish pink because they absorb at shorter wavelengths. The

rather simple addition of one methyl substituent to a carbon atom usually has

(in terms of dye chemistry) only a small bathochromic effect, i.e. the shift of

the absorptions to longer wavelengths are small; it is directly correlated to the

inductive effect of the methyl group, which is also relatively small. Conse-

quently, the addition of a methyl substituent alone cannot explain the striking

difference in colour between tertiary S–nitrosothiols and their less substituted

analogues. This consideration could have been the basis for structured re-

search, even though the compounds are less stable and more difficult to han-

dle.

In 1999, Aurlsamy et al. calculated the energy of activation of the syn-anti-

rotation to be at 53.8 kJ mol%1 (12.7 kcal mol%1) for S-nitrosomethylthiol and

Chapter 7

96

measured 44.8 kJ mol%1 (10.7 kcal mol%1) for the same process in S-nitroso-

triphenylmethylthiol ((C6H5)3CSNO) [38]. Furthermore, they found that their

calculations at the B3LYP/6-311+G* level cannot satisfyingly reproduce the

experimental findings: for example the S–N bond was overestimated in their

calculations by 10 pm, which means that considerable stabilisation of that

bond by delocalisation (or resonance) takes place. They observed that S–

nitrosoacetyl-penicillamine is stable in light, but S-nitroso-

triphenylmethylthiol is not, although both are compounds that contain a qua-

ternary carbon atom next to the sulfur atom. Usually, these are more stable

than their less substituted derivatives. The photosensitivity of S-nitroso-

triphenylmethylthiol is unexpected. Aurlsamy et al. did compare the crystal-

lographic data of that compound with the data known for S–nitrosoacetyl-

penicillamine and stated that they were “very similar”. Unfortunately, the an-

gles around the quaternary carbon atom were not compared, although they

were known: in S–nitrosoacetyl-penicillamine the carbon substituents adopt

angles of 103°, 108° and 111° relative to the sulfur atom. In S-nitroso-

triphenylmethylthiol the base of the tetrahedron is flattened, i.e. three angles

of 102°, which is a consequence of a very stable resonance state, this mole-

cule can adopt: the triphenylmethylium-ion ((C6H5)3C+), which is why S-

nitroso-triphenylmethylthiol might show significantly different characteristics

in reactivity.

It was only in 2000 that this matter was re-investigated by Bartberger et. al.

[119] with compounds that contained primary to quaternary carbon atoms at

the position proximal to the sulfur atom: the study confirmed that the com-

pounds with quaternary carbon atoms preferred a anti conformation, but it

revealed also that for the less substituted carbon atoms the syn conformation

was preferred. The same study also found, that the energy of activation for

syn-anti rotation is smaller, hence this rotation is possible for some com-

pounds at room temperature. Furthermore, calculations predicted +CSN an-


97

gles of 102 – 109° for the syn type molecule, whereas in the anti type the

same angle is between 94° and 97°. The S–N bond lengths of the anti-

conformers were slightly longer (e.g. 1.88 pm instead of 182 pm) in the same

calculations. The increase in S–N bond length is most pronounced in the

compound with the quaternary carbon atom. All other calculated geometrical

values match those from an earlier study [38]. However, the differences in

Gibbs energies of the rotamers differ: Bartberger et. al. obtained values that

were one magnitude higher, i.e. 4 kJ mol%1 (1 kcal mol%1) instead of

0.4 kJ mol%1 (0.1 kcal mol%1). Particularly the compound with the quaternary

carbon atom preferred the anti conformation by a factor of five, in which both

studies agree.

This refinement to vary the number of substituents at the carbon atom next to

the S-nitroso group is most important: the S–nitrosothiols in human physiolo-

gy are derivatives of cysteinyl residues in peptides and proteins, and the car-

bon atom proximal to the sulfur atom is a primary one. Reliable results of

structurally related compounds with syn conformation replaced the rather

broad generalization of the 1990 decades favouring the anti conformer with a

relatively high activation barrier.

These facts can also explain the differences between the quaternary carbon

type compounds and the less substituted type carbon (Fig. 7.3):

• The visible absorption was assigned to the nN * (SN* transition [35].

• The molecule can only adopt syn configuration, if the carbon atom

proximal to the sulfur atom carries three or less substituents, otherwise

the steric hindrance is too large.

• When the molecule is in syn configuration, electron density can be

transferred from the nO orbital to the (SN* orbital by negative hypercon-

jugation [110], which stretches the S–N bond and changes the absorp-

Chapter 7

98

tion behaviour, which is in agreement with the increased bond length

calculated for such a molecule.

• Consequently, the bond angle +CSN is rather towards 104° which is

characteristic for water molecules, where the oxygen atom is hybridised

but the nonbonding electron pairs occupy more space than the bonds to

the hydrogen atoms.

• The negative hyperconjugation destabilizes the bond between the sulfur

and the nitrogen atom, which is why less substituted type compounds,

which adopt syn configuration, dissociate faster to thiyl radicals and ox-

idonitrogen(•) then the quaternary carbon type adopting anti configura-

tion is virtually stable.

• In the anti configuration, the nS orbital hinders the oxygen atom to

adopt a position that allows interaction with the other nonbonding or-

bital of the oxygen atom n’O. This avoids the bond lengthening and a

geometry of an approximately 90° bond angle (+CSN) typical for sul-

fur is established.

Figure 7.3: A) The negative hyperconjugation between the oxygen and the nitrogen atom. B) A syn configuration for qua-ternary carbon type molecules is unfavourable. C) Due to the nonbonding electron pair of the sulfur atom, the oxygen atom cannot adopt a geometry that allows hyperconjugation and all destabilizing effects are omitted.


99

The kind of model compounds presented by Bartberger et al. in 2000 [119]

might be not apply to S–nitrosoglutathione: although S–nitrosoglutathione

belongs to the group of compounds with a secondary carbon atom adjacent to

the sulfur atom, the longer chain differs a great deal from the methyl group

discussed in that study.

In order to get closer to the structural implications on reactivity of glutathione

and cysteine, this chapter presents some results of investigations on S–nitroso-

tert-butylthiol and S–nitroso-neo-pentylthiol and show that extrapolations

from model molecules can be misleading and that there must be additional

structural features than the ones unveiled so far.

Chapter 7

100

7.2 Results

Visible Spectroscopy

Although visible and ultraviolet spectroscopy is a rather facile method, many

investigations relied on modern techniques as nuclear magnetic resonance

(NMR). The visible spectra of many S–nitroso compounds possess an asym-

metric band in the region of 600 nm for compounds of the type with quater-

nary carbon atoms and in the region of 540 nm.

The spectra of this region of S–nitroso-tert-butylthiol in various organic sol-

vents (Fig. 7.4) shows that the absorption band shows a fine structure tenta-

tively attributed to the syn/anti conformation change that dominates the dis-

cussion about the structural features of S–nitrosothiols.

Figure 7.4: Spectra of S–nitroso-tert-butylthiol in hexane (solid, black), acetonitrile (dotted, grey) and methanol (dashed, grey) from 500 to 650 nm that shows the visible band. All spectra show three main absorption peaks at approx. 520 nm, 560 nm and 600 nm. The spectrum in hexane reveals the underlying structure of at least two maxima in every peak.

In order to test the hypothesis that the reason for this fine structure is closely

related with the nitrogen atom in the –SNO group, the spectra of S–nitroso-

tert-butylthiol synthesised with and without [15N]-sodium dioxidonitrate

(Na15NO2) were determined. These spectra were recorded in hexane, because

this solvent resolves the fine structure best (Fig. 7.5).

$ hexane % acetonitrile & methanol

wavelength / nm

arbi

trary

abs

orba

nce

units


101

Figu

re 7

.5:

The

spec

trum

of

S–n

itros

o-te

rt-bu

tylth

iol (

dash

ed,

blac

k) a

nd o

f [15

N]-S

–nitr

oso-

tert-

buty

lthio

l (so

lid,

grey

, th

e m

axim

al a

bsor

ptio

n w

as m

atch

ed t

o th

e sp

ectru

m o

f th

e no

n-is

otop

e la

belle

d co

mpo

und)

. Th

e sp

ectra

l fe

atur

es c

oinc

ide

at a

ppro

x. 5

80 n

m,

the

[15N

]-va

riant

sho

ws

long

er p

eak-

to-p

eak

dist

ance

s, it

s re

lativ

e m

axim

a ar

e sh

ifted

to

low

er w

ave-

leng

ths

belo

w 5

80 n

m, a

nd to

long

er w

avel

engt

h ab

ove

the

sam

e w

avel

engt

h.

$

15N

%

14

N

Chapter 7

102

Figure 7.6: Spectrum of gaseous S–nitroso-tert-butylthiol at different temperatures normed to the maximum value in the re-gion selected. At higher temperatures the peak ratios are clos-er to 1:1 than lower temperatures. The arrow colours designate peak series. Evaluating the values for the peaks between 605 and 580 nm with ,G = !R·T·ln K yields a Gibbs energy differ-ence between black and gray states of 0.36±0.04 kJ mol!1 (or 0.01 kcal mol!1) and for black and white states approximatively 1.7±0.04 kJ mol!1 (or 0.4 kcal mol!1) (see Fig. 7.12).

A clear shift of the spectra confirms the connection of the band and its fea-

tures to the nitrogen atom, as expected from the spectral assignment found in

the literature [35]. The same absorption phenomenon in the spectrum of S–

nitroso-neo-pentylthiol is far less pronounced, i.e. the ratio between the main

and the second maximum in the spectral region discussed is approx. 2:1

(Fig. 7.7), whereas for S–nitroso-tert-butylthiol it was 6:1. In contrary to the

previous compound, he spectrum does not show a third peak in the region of

520 nm.

wavelength / nm

arbi

trary

abs

orba

nce

units


103

Figure 7.7: Spectra of an aqueous solution of S–nitroso-neo-pentylthiol at concentrations of 1 mM (black) and 10 "M (grey, 10-fold enlarged). The peak at 590 nm does not show fine structure, therefore the underlying ground states must be either close in energy so they cannot be distinguished, or they are largely separated and there is only one that is populated.

Nuclear Magnetic Resonance

As S–nitroso-tert-butylthiol has already been intensively investigated in terms

of conformation isomerism, energies of activation and related features (as

shown in the introduction), the determination of the energy of activation of

the syn/anti conformational change of S–nitroso-neo-pentylthiol was of inter-

est.

Chapter 7

104

Figu

re 7

.8:

Inte

rmed

iate

mag

nifi-

catio

n of

the

coal

es-

cenc

e m

easu

rem

ents

of

S–n

itros

o-ne

o-pe

ntyl

thio

l in

step

s of

20

K (o

ther

mea

sure

-m

ents

wer

e om

itted

for

clar

ity).

Gre

y ar

row

s m

ark

coal

esci

ng p

eaks

of

the

233

K s

pect

rum

, bl

ack

arro

ws

the

ones

of

the

313

K s

pect

rum

. Th

e w

hite

arr

ow p

oint

s to

the

seco

nd b

road

en-

ing

that

indi

cate

s th

at

two

coal

esce

nce

pro-

cess

es m

ay o

ccur

. The

st

ars

mar

k im

purit

ies

due

to th

e co

rres

pond

-in

g th

iol.


105

Figure 7.9: Enlargement of the region between 0.5 and 1.5 ppm in Fig. 7.8. Small shifts of the impurity signals (marked with stars) along the x-axis are due to the temperature de-pendence of the resonance. The unchanged shapes of the sig-nals indicate that they are not part of the coalescence system. The small signals at 0.8 and 1.11 ppm are satellites of the large signal at 0.95 ppm caused by the small number of 13C at-oms present.

The 1H-NMR recordings of S–nitroso-neo-pentylthiol at intervals of 10 K

(Figs. 7.8 and 7.9, overview Fig. 7.11) show coalescence of multiple signals

along with impurity signals of neo-pentylthiol and the corresponding disul-

fide. A simple coalescence sequence over a series of temperatures or frequen-

cies usually shows two individual peaks that broaden out and merge together

giving rise to one unified signal. This is due to a group of atoms in a molecule

that rotates or flips at relatively low frequencies. A low measuring tempera-

ture causes this group to move at lower frequencies (i.e. slower) than the time

resolution of the instrument and allows resolving the signals of individual hy-

drogen atoms. Inversely, a high measuring temperature accelerates the

movement and the instrument only detects a mean signal of all hydrogen at-

oms concerned.

Chapter 7

106

Figure 7.10: Arrhenius plot and linear fit of the coalescence sequence. The slope represents the energy of activation of ap-prox. 50 kJ mol!1 (12 kcal mol!1) in units of !R (8.31 J mol!1 K!1). The inverse temperature dependence is better described by a curve, which might be caused by the two superimposed coalescence phenomena suspected.

The evaluation of the relative integrals yields the equilibrium constant at the

temperature measured. From an Arrhenius plot (Fig. 7.10) an approximate

energy of activation of approximately 50 kJ mol%1 (12 kcal mol%1) is derived.

The data do not correspond to an ideal Arrhenius type reaction, because they

are better described by a curve.

Closer inspection of the coalescence sequence reveals an atypical behaviour:

from low to high temperature several signals disappear but more than one sig-

nal re-appears: no clear attribution of n peaks at low temperatures to one peak

at high temperature is possible. Additionally, instead of one broadening and

the accompanying loss of intensity, two broadenings can be seen, which indi-

cates two overlapping coalescences and, equivalently, two groups of atoms

that start moving in the temperature range measured.

ln(K

)


107

Figu

re 7

.11:

Ove

rvie

w o

f

the

spec

trum

Chapter 7

108

7.3 Discussion

The UV-Vis measurements of the vapour of S–nitroso-tert-butylthiol exhibit a

repeating pattern of three overlapping peaks: the first sequence is at 605, 590

and 580 nm. This pattern is repeated three times. The peak shapes suggest that

the first (sharp, 605, 560, 525 nm), second (diffuse) and third peaks of every

pattern belong to the same vibronic state and represents transitions from the

ground state to the first three excited states. So at least twelve states are in-

volved in this band (Fig. 7.12). Rotatory states can be excluded as a reason,

because their energy differences lie in the region of microwaves. f-orbital

transitions also show very sharp peaks, but with a molecule that consists of

atoms of the second and third row in the periodic table, this can also be ruled

out.

Figure 7.12: Schematic representation of the vibronic states found in the visible absorption band of S–nitroso-tert-butylthiol (not to scale).

The order of magnitude of the energy differences of the states v0,0 and v1,0 For

S–nitroso-tert-butylthiol matches the calculated ones [119]. Still, the meas-

ured values for S–nitroso-tert-butylthiol represent two different changes in

conformation: the rotation of the trimethylmethyl group (tentatively assigned

to the difference of 0.36±0.04 kJ mol%1 or 0.01 kcal mol%1) and the syn-anti-


109

rotation of the –NO group (tentatively assigned to the difference of

1.7±0.04 kJ mol%1 or 0.4 kcal mol%1). The value for the syn-anti-rotation lies

in between the values of the studies discussed in the introcuction [119; 38].

The UV-Vis spectrum of S–nitroso-neo-pentylthiol cannot be evaluated in the

same way. The width of the peaks suggests that the vibronic ground states lie

close together rather than far apart and that the energy difference between the

ground states is smaller than in the case of S–nitroso-tert-butylthiol. There are

no calculations available for this molecule, but calculations for similar com-

pounds in the same study [119] confirm this tendency. This supports the mod-

el in which the tert-butyl group hinders syn-anti-rotation: the further away the

tert-butyl group is from the –SNO group the smaller the energy difference.

Furthermore, the peaks in this spectrum lie closer together, which means that

the energy differences between the ground state and the excited states are also

smaller, i.e. the molecule moves or shakes more easily.

The energy differences for S–nitroso-neo-pentylthiol from the coalescence

series are 5.1±0.2 kJ mol%1 (1.2 kcal mol%1) for the difference of the ground

states (vx,0) agreeing roughly with Bartberger et al. and 54±2 kJ mol%1

(12 kcal mol%1) for the energy of activation, which is in good agreement with

Aurlsamy et al.. These values are larger than the energy differences computed

and estimated for S–nitroso-tert-butylthiol from the UV-Vis: This strongly

suggests, that there are more structure-reactivity effects acting on cysteine-

like structures than discussed so far in the literature. There are not many pos-

sible causes for that: The effects of the S-nitroso group have been rather ex-

tensively studied, as presented in chapter 7.1. The tert-butyl group is main-

tained in both compounds investigated in the present study. This leaves the

possibility that there are more interactions between the sulfur atom and its

proximal carbon atom. This could comprise charge transfer mechanisms as

well as further hyperconjugation: either a second, independent hyperconjuga-

Chapter 7

110

tion system acts between the sulfur atom and the proximal carbon atom and

its substituents, or a second hyperconjugation system that interacts with the

nitrosyl group or with any further functionality of the sulfur atom, e.g. as a

thiyl radical. The increase in S–N bond length being most pronounced in the

compound with the quaternary carbon atom [119] supports an interaction be-

tween the sulfur atom and the carbon atom next to it (Fig 7.13):

Figure 7.13: Schematic representations of linear combinations of atomic orbitals in an RSNO compound. The transition nN!(*SN causes the typical visible absorption. The (*SN orbital can interact with the (*CH orbital. In Fig. 7.3 A the (*SN orbital shows another possible interaction with the nO oribtal. Consid-ering all these interactions, the reactivity should be judged ra-ther in terms of a –CSNO group rather than a –SNO group.

To support this, Everett et al. [22] produced radicals form cysteine disulfide

and penicillamine disulfide with laser flash photolysis. These two compounds

do not yield the same products when laser flash photolysis is applied under

the same conditions: the cysteine disulfide yields a twofold excess of thiyl

radicals (CyS•) over perthiyl radicals (CySS•), whereas with penicillamine

disulfide four times more perthiyl radicals (PenSS•) than thiyl radicals (PenS•)


111

are found. So it is evident that the carbon atom proximal to the sulfur atom

holds a key position in reactivity.

Unfortunately, although the coalescence measurements were performed over

the full temperature range of the instrument, the full range of coalescence

phenomena could not be observed: at the highest temperature, the peaks did

not shift to their final position at high temperature. This prevents a clear as-

signment of the peaks to defined structures as well as a closer understanding

of the underlying processes. The Arrhenius plot (Fig. 7.10) is therefore not

completely linear, but exhibits a small curvature.

It seems that the relation between structure and reactivity of cysteine still

holds some open questions that need answers before we are able to fully un-

derstand the role of thiyl radicals, S–nitrosothiols and thiols in physiology and

chemistry.

Chapter 7

112

7.4 Acknowledgement

This work would not have been possible with the expertise and know how of

Heinz Rüegger (†) of the NMR facilities of the Institute for Inorganic Chem-

istry at ETH Zurich.

This work was supported by the ETH Zurich and the Swiss National Science

Foundation.

Chapter 8

General Conclusion and Perspectives

Chapter 8

114

8 General Conclusion and Perspectives

The thiyl radicals in general and the glutathione thiyl radical in particular

have been long believed to be mostly harmless. They were known mostly in

biochemistry, and their role in physiological processes seemed clear. Also,

biochemistry, which boosted in the last century, focused rather on metabolic

steps, rather than detailed mechanisms and time resolution.

The thiyl radicals slid only very slowly in the focus of bio-inorganic and

physical chemists, who gained more and more interest in the interesting and

complex features that these molecules have to offer. Unsure of the meaning of

the results in the beginning, the entanglement, i.e. H-abstraction, between C-

and S-centred radicals was discovered. Of course, as soon as new C–C bonds

can be formed from C-centred radicals emerging from the thiyl radicals, the

latter are not to be regarded mostly harmless anymore.

It is no surprise, that we can show, that the glutathione thiyl radical also fol-

lows that pattern. Nevertheless, the manner in which it does is rather surpris-

ing: against intuition the proportion of C-centred radicals is rather high and

there are not two species participating in this equilibrium but at least three.

Although UV-Vis spectroscopy cannot pinpoint the locations the H–

abstraction takes place, it clearly indicates that there must be at least two dif-

ferent sites, suggesting !C- and "C-centred radicals.

This can be confirmed and extended with NMR measurements of experiments

in deuterated water, where an exchange of H by D is clearly measureable.

They also show, that the range of active sites for H-exchange is wide, seem-

ingly unpredictable and highly dependent on the peptide composition.

Although the connection between S- and C-centred radicals is known for ap-

proximately two decades, the news have not spread that the thiyl radicals are

not mostly harmless, but have a strong protein damage and therefore ageing

General Conclusion and Perspectives

115

potential. The new face of the thiyl radicals must find its way back to the bio-

chemists and to all other fields of science that are involved with this species.

Furthermore the NMR experiments show, that the connection between ab-

straction site and peptide structure is in complete oblivion and that a whole

new field must be seized and understood in further experiments.

That oxidonitrogen(•) does not or not sufficiently fast react with the thiyl rad-

ical is than not surprising either, the distribution between C- and S-centred

radicals reduces crucial S-centred radical concentration for the reaction with

oxidonitrogen(•).

The structural influence of simple methyl groups is strong and clear. Never-

theless, the closer connection between structure and reactivity is not under-

stood yet. This is partly because most of the interest and therefore most of the

research are focused on the redox-properties of S–nitroso compounds, i.e.

whether these compounds generate free radicals, how fast and in what form,

particularly in combination with large fields of research, such as oxidonitro-

gen(•) or anti-oxidants in ageing.

But the neglect of structural features and their influence on reactivity might be

the key to some crucial advancement: many results of reactions of thiols and

their derivatives in the free radical context are not fully understood, but rather

accepted, e.g. the inverse distribution of thiyl and perthiyl radicals of the pho-

tolysis of cysteine disulfide or penicillamine disulfide. Further work in the

field of inorganic or even physical chemistry is needed.

Sulfur is a very polyvalent atom, in both senses, and so are the possibilities of

the reactions involving sulfur-containing peptides. This multitude of reactivi-

ties of sulfur was also encountered during this work, and it shows, that this

field still has many challenges to offer and is worth being unveiled.

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CURRICULUM VITAE

Dustin René Hofstetter Born on 21st December 1977

Citizen of Steinhof (SO), Switzerland Education 1998 Matura Typus C (mathematics and science) at the

Kantonsschule Zug, Zug, Switzerland 1999 – 2004 Studies in Chemistry at ETH Zurich, Zurich, Switzerland 2003 – 2004 Diploma Thesis in analytical chemistry at ETH Zurich at

the Department of Chemistry and applied Biosciences with Prof. Dr. Ern2 Pretsch on The Automatic NMR-Spectra Comparison.

2004 – 2012 Doctoral Thesis at ETH Zurich in the Department of Chemistry and applied Biosciences with Prof. Dr. Willem H. Koppenol on The Glutathione Thiyl Radical.

2011 – to date Studies in Chemical Education at ETH Zurich Professional experience 1998 – 2002 Programmer and Customer Advisor for Databases

(FileMaker) for MacRobi GmbH, Rotkreuz 2005 – 2010 Teaching Assistant in exercise and practical courses 2005 – 2008 House Responsible for Woko Genossenschaft (housing),

Zürich 2010 – to date Teacher for Chemistry at the Kantonsschule Zug Further experience 1998 – 2005 Member of various boards of student organisations 2002 – 2008 Formation and work as a specialist for chemical warfare

for the Swiss Army 2005 – 2010 President of the Swiss Chemistry Olympiads Association

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