abstract gas-phase chemistry of tryptophan … · 50 2.8 comparison of experimental irmpd (black...

ABSTRACT

GAS-PHASE CHEMISTRY OF TRYPTOPHAN-BASED RADICALS

Andrii Piatkivskyi, Ph.D.

Department of Chemistry and Biochemistry

Northern Illinois University, 2014

Victor Ryzhov, Director

This work is devoted to the fundamental study of gas-phase tryptophan-based

radical cations. It covers: mechanisms of the radical ion formation in the gas-phase; the

description and contrast of the two types of tryptophan side chain radicals (π and

N-indolyl) based on their reactivities, infrared spectra, structural energetics and

fragmentation patterns; investigation of the N-indolyl tryptophan radical cation

fragmentation pathways; formation of the two types of tryptophan radicals (π and

N-indolyl) within short peptides; their characterization and comparison based on the

fragmentation patterns and reactivity; and gas-phase study of intramolecular radical

migration from tryptophan to cysteine and from tryptophan to tyrosine side chains within

short peptides.

Two different approaches were followed to regiospecifically form desired radicals

on the tryptophan side chain. The tryptophan π-radical cation was formed via electron

transfer during dissociation of ternary metal complex ([CuII(terpy)(Trp)]

2+), while

N-indolyl radical was formed by the homolytic cleavage of the NO group from the

N-nitrosylated tryptophan during gas-phase fragmentation. Both radicals were exposed to

the low-energy collision-induced dissociation in order to elucidate and contrast their

fragmentation. To estimate the fragmentation pathway of the N-indolyl tryptophan

radical cation, additional experiments (including H/D exchange, dissociation of

tryptophan derivatives and DFT calculations) were carried out. The radical reactivity has

been tested via gas-phase ion-molecule reactions with benzeneselenol, 1-propanethiol and

di-tert-butyl nitroxide. The π-radical cation was found to be more reactive compared to

the N-indolyl radical, which was explained by the charge presence near the radical site.

Propanethiol was found to be reactive towards π-radical cation and showed the lack of

reactivity with N-indolyl radical. To investigate the role of amino acid sequence, both

types of the radicals were formed within short peptides (AW, WA, GW, WG, GGW).

They were also contrasted by their fragmentation and reactivity. The gas-phase radical

migration between tryptophan and cysteine (CW, CGW, CGGW model peptides), and

tryptophan and tyrosine (GYW, YGW, WGY model peptides) side chains was examined

at thermal conditions and with application of additional collision energy. Radical

migration between N-indolyl tryptophan radical and tyrosine side chains has been

observed. Investigation of the migration between tryptophan π-radical cation and cysteine

side chain remains to be work in progress.

NORTHERN ILLINOIS UNIVERSITY

DE KALB, ILLINOIS

DECEMBER 2014

GAS-PHASE CHEMISTRY OF TRYPTOPHAN-BASED RADICALS

BY ANDRII PIATKIVSKYI

© 2014 Andrii Piatkivskyi

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE

DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

Doctoral Director:

Victor Ryzhov

ACKNOWLEDGEMENTS

This dissertation would be impossible without my brother, Dr. Yuriy Pyatkivskyy,

who inspired me to apply to graduate school by his own example, and my family who

supported me in my good and bad times.

My sincere gratitude to my committee members who made a huge impact in my

graduate school life: Dr. David Ballantine, who made a significant influence on me at the

very beginning of my way; Dr. Chong Zheng, Dr. Lee Sunderlin, Dr. James Horn and

Dr. Martin Kocanda for their help, inspiring talks and bright ideas; and my adviser

Dr. Victor Ryzhov who was a great teacher and mentor with limitless patience. Also I

would like to thank all the past and present members of Dr. Ryzhov's research group,

especially Dr. Sandra Osborn and our collaborators, Dr. Alan Hopkinson and Dr. Justin

Kai-Chi Lau, from York University, Toronto, Canada, who supported our experimental

data with theoretical calculations. It was a great pleasure to work together with you.

Special thanks to Ms. Patricia Austin from the NIU Department of English.

Many thanks to all NIU Department Chemistry and Biochemistry faculty and staff

for their support and help. Thank you for a chance to learn, to grow as a professional and

a person, and to meet with people I would never have met otherwise.

DEDICATION

To M.D. Nicholas Cary, it was a great honor to know you.

iv

TABLE OF CONTENTS

Page

LIST OF FIGURES ........................................................................................................... vii

LIST OF TABLES ............................................................................................................ x

LIST OF SCHEMES........................................................................................................ xi

LIST OF APPENDICES ...................................................................................................xiii

LIST OF ABBREVIATIONS ...........................................................................................xiv

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

2. STRUCTURE AND REACTIVITY OF THE DISTONIC AND AROMATIC

RADICAL CATIONS OF TRYPTOPHAN ..................................................................... 30

2.1 Introduction ............................................................................................................. 30

2.2 Experimental Section .............................................................................................. 35

2.2.1 Chemicals and Reagents ................................................................................... 35

2.2.2 Mass Spectrometry: CID and Ion-Molecule Reactions .................................... 35

2.2.3 Infrared Multiple Photon Dissociation Spectroscopy ...................................... 36

2.2.4 Theoretical Calculations ...................................................................................... 37

2.3 Results and Discussion ............................................................................................ 39

2.3.1 Collision-Induced Dissociation ........................................................................ 39

2.3.2 Ion-Molecule Reactions .................................................................................... 42

2.3.3. Theoretical Calculations ................................................................................. 47

2.3.4. Infrared Multiple Photon Dissociation ............................................................ 50

2.3 Conclusions ............................................................................................................. 59

v

Chapter Page

3. INVESTIGATION OF FRAGMENTATION OF TRYPTOPHAN NITROGEN

RADICAL CATION ......................................................................................................... 63

3.1 Introduction ............................................................................................................. 63



3.2.2 Mass Spectrometry ........................................................................................... 63


3.3.1 Collision-Induced Dissociation ........................................................................ 64

3.3.2. Theoretical Calculations ................................................................................. 73

3.4 Conclusions ............................................................................................................. 78

4. STRUCTURE AND REACTIVITY OF THE DISTONIC AND AROMATIC

RADICAL CATIONS OF TRYPTOPHAN-CONTAINING PEPTIDES ....................... 80

4.1 Introduction ............................................................................................................. 80



4.2.2 Mass Spectrometry: CID and Ion-Molecule Rreactions .................................. 90

4.2.3 Infrared Multiple Photon Dissociation Spectroscopy ...................................... 91


4.3.1 Collision-Induced Dissociation and Ion-Molecule Reactions .......................... 92

4.3.2. Conclusions ................................................................................................... 102

5. GAS-PHASE STUDY OF INTRAMOLECULAR RADICAL MIGRATION OF

TRYPTOPHAN-BASED RADICALS WITHIN SHORT PEPTIDES .......................... 104

5.1 Introduction ........................................................................................................... 104

5.2 Study of Tryptophan-to-Cysteine Side-Chain Radical Migration......................... 106

5.2.1 Experimental Section ......................................................................................... 108

5.2.1.1 Chemicals and Reagents .............................................................................. 108

vi

Chapter Page

5.2.1.2 Mass Spectrometry: CID and Ion-Molecule Reactions ............................... 108

5.2.2 Results and Discussion ....................................................................................... 109

5.2.2.1 Collision-Induced Dissociation ................................................................... 109

5.3 Study of Tryptophan-to-Tyrosine Side-Chain Radical Migration Within Short

Peptides ....................................................................................................................... 116

5.3.1 Experimental Section ......................................................................................... 116

5.3.1.1 Chemicals and Reagents .............................................................................. 116

5.3.1.2 Mass Spectrometry: CID and Ion-Molecule Reactions ............................... 117

5.3.2 Results and Discussion ....................................................................................... 118

5.3.2.1 Collision-Induced Dissociation ................................................................... 118

5.4 Conclusions and Future Work ............................................................................... 128

6. CONCLUSIONS......................................................................................................... 109

6.1 Future Work .......................................................................................................... 139

REFERENCES ............................................................................................................... 136

APPENDICES ................................................................................................................ 165

LIST OF FIGURES

Figure Page

2.1 CID spectra of: tryptophan radical cation formed via CID of

[CuII(terpy)(Trp)]

•2+ (eq. 1) (top panel); tryptophan radical cation

formed via CID of H3N+-TrpN-NO (eq. 2) (middle panel);

protonated tryptophan (bottom panel)................................................ 40

2.2 B3LYP/6-311++G(d,p)-calculated reaction pathways for the

rearrangement of 2 into 1.................................................................... 41

2.3 Ion-molecule reaction spectra of 1(top) and 2 (bottom) with

benzeneselenol. Neutral benzeneselenol was delivered via pulsed

valve, reaction time 200 ms (top) and 2000 ms (bottom)................... 45

2.4 Ion-molecule reaction spectra of 1 (top) and 2 (bottom) with

di-tert-butyl nitroxide. Neutral di-tert-butyl nitroxide was delivered

via pulsed valve, reaction time 200 ms (top) and 2000 ms

(bottom)............................................................................................... 46

2.5 Ion-molecule reaction spectra of 1 (top) and 2 (bottom) with

n-propyl thiol. Neutral n-propyl thiol was delivered via pulsed

valve, reaction time 50 ms (top) and 2000 ms (bottom)..................... 47

2.6 B3LYP/6-311++G(d,p)-calculated structures, energies, and electron

spin densities (bold number) of indolyl nitrogen s radical (left) and

indolyl π-radical (right)....................................................................... 48

2.7 B3LYP/6-311++G(d,p)-calculated structures and spin densities of

Trp•+

π-radical 1 and NH3+TrpN

• nitrogen radical 2........................... 50

2.8 Comparison of experimental IRMPD (black trace) of tryptophan

radical cation formed via SORI-CID of [CuII(terpy)(Trp)]

•2+ (eq. 1)

and DFT-calculated (red trace) IR spectra of 1, 2, 3, and 4................ 52

viii

Figure Page


radical cation formed via in-source CID of H3N+-TrpN-NO

+ (eq. 2)

and DFT-calculated (red trace) IR spectra of 1, 2, 3, and 4................ 53


radical cation formed via SORI-CID of [CuII(terpy)(Trp)]

•2+ (eq. 1)

and DFT-calculated (red trace) IR spectra of four low-energy

conformers of 1................................................................................... 57


radical cation formed via in-source CID of H3N+-TrpN-NO

+ (eq. 2)

and DFT-calculated (red trace) IR spectra of four low-energy

conformers of 2................................................................................... 58

3.1 Fragmentation spectra of tryptophan NH3+TrpN

•............................... 65

3.2 Ion-molecule reactions of m/z 131 ions left panel H3N+-TrpN

fragment, right panel 3-methylindole radical with di-tert-butyl

nitroxide.............................................................................................. 67

3.3 Fragmentation spectrum of N-acetyl-L-tryptophanamide.................. 69

3.4 Fragmentation spectrum of L-tryptophan methyl ester....................... 70

3.5 Fragmentation spectrum of D3N+-TrpN•............................................. 73

3.6 Tryptophan NH3+TrpN• radical fragmentation path calculations........ 76

3.7 Fragmentation spectrum of tryptamine N-radical cation.................... 77

4.1 CID spectrum of [CuII(terpy)WA]2+ complex.................................... 93

4.2 CID spectrum of the N-nitrosylated WA peptide............................... 95

4.3 CID spectra of nWA•+

radical (top panel) and nWA*•+

radical withdeuterium-labeled alanine side chain.................................................. 96

ix

Figure Page

4.4 CID spectrum of nAW•+

radical.......................................................... 98

4.5 CID spectrum of πAW•+

radical.......................................................... 99

4.6 Ion-molecule reaction of πAW•+

with n-propyl thiol.......................... 100

5.1 Amino acid side chain radical interconversion................................... 105

5.2 Intermolecular formation of S-based radical via PCET of the

tryptophan π-radical cation (a), intramolecular formation of S-based

radical via tryptophan-to-cysteine side-chain radical

migration (b)....................................................................................... 107

5.3 Mass spectra of a) ternary metal complex of CGW, b) CID of

[CuII(terpy)CGW]

•2+ ion..................................................................... 110

5.4 CID spectra of a) m/z 307 ion formed from CW peptide, and

b) m/z 364 ion formed from CGW peptide......................................... 111

5.5 CID spectra of a) nCW•+

, b) CGW•+

, and c) CGGW•+

........................ 114

5.6 Ion-molecule reaction of nCGW•+

with dimethyl disulfide

a) no collision energy applied, b) with collision energy..................... 115

5.7 Comparison and contrast of CID fragments of a) nGW•Y

+,

b) nGYW•+

and c) nYGW•+

radical cations......................................... 122

5.8 CID of the metal ternary complex with formation of a) GW•Y

+,

b) GYW•+

, c) YGW•+

(all m/z 424) and G•GW

+ (m/z 318)

radicals................................................................................................ 124

5.9 Comparison and contrast of CID fragments of a) GW•Y

+,

b) GYW•+

, c) YGW•+

radicals, produced via dissociation of copper

ternary complexes............................................................................... 125

vii

LIST OF TABLES

Table Page

1.1 Rate constants of intramolecular radical reactions of between

tryptophan and tyrosine within a peptide............................................... 15

4.1 Comparison and contrast of CID fragments of protonated GGW,

nGGW•+

, πGGW•+

and G•GW

+ radicals................................................ 89

4.2 Comparison and contrast of CID fragments of protonated WA and

nWA radical........................................................................................... 97

4.3 Comparison and contrast of CID fragments of protonated WA and

nWA radical........................................................................................... 101

5.1 Comparison of CID fragments of nCW•+

, nCGW•+

, nCGGW•+

radicals................................................................................................... 113

5.2 Comparison and contrast of CID fragments nGYW•+

and nYGW•+

,

nGW•Y

+ radicals.................................................................................... 120

5.3 Comparison and contrast of CID fragments of GYW•+

, YGW•+

, and

GWY•+

radicals, formed via ternary complex dissociation................... 127

viii

LIST OF SCHEMES

Scheme Page

1.1 Glycyl radical...................................................................................... 3

1.2 The radical transfer via hydrogen atom exchange between glycine

and cysteine......................................................................................... 7

1.3 Tyrosyl radical.................................................................................... 7

1.4 Tryptophan radical cation (a) and tryptophan neutral nitrogen

radical (b)............................................................................................ 11

1.5 Examples of hydrogen-rich and hydrogen-deficient radicals............. 21

1.6 Typical dissociations of hydrogen-rich peptide cation radicals upon

ExD..................................................................................................... 22

1.7 Typical dissociations of hydrogen-deficient amino acid radicals via

radical migration................................................................................. 23

1.8 Cβ–Cγ bond cleavage of tryptophan side chain by homolytic

aromatic substitution reaction............................................................. 26

2.1 Tryptophan radical cation (a) and tryptophan neutral nitrogen

radical (b) in proteins.......................................................................... 31

2.2 Low-energy isomers of tryptophan radical cations: π-radical (1),

indolyl nitrogen radical (2), α-carbon radical (3), and β-carbon

radical (4)............................................................................................ 33

3.1 Possible ways of forming 3-methylindole radical cation from

different molecules.............................................................................. 66

3.2 Structures of tryptophan derivatives used to examine the proposed

hydrogen transfers............................................................................... 68

xii

Scheme Page

3.3 Structure of D3N+-TrpN

•..................................................................... 71

3.4 Positions of the hydrogens involved in the fragmentation process,

determinate during H/D exchange experiment................................... 72

3.5 Calculated isomerization energy barrier for tryptophan radicals........ 74

3.6 Hydrogen atoms transfer within tryptophan nitrogen radical during

fragmentation....................................................................................... 75

4.1 Structures of a) protonated GGW, b) nGGW•+

, c) πGGW•+

d) G•GW

+ radicals............................................................................... 86

4.2 Structures of a) πAW•+

, b) nAW•+

radicals......................................... 88

4.3 Structures of a) πWA•+

, b) nWA•+

radicals......................................... 94

5.1 The mechanism of the radical migration between tryptophan and

tyrosine side chains............................................................................. 119

5.2 Experiments outlining tryptophan-to-tyrosine radical migration

investigation via CID........................................................................... 119

x

LIST OF APPENDICES

Appendix Page

A. CONTROL MASS SPECTRA FOR THE AW AND WA

PEPTIDES................................................................................. 165

B. IRMPD SPECTRA FOR THE nAW AND nWA PEPTIDE

RADICALS................................................................................ 167

C. CID MASS SPECTRA OF GW, WG AND GGW RADICAL

CATIONS AND CONTROLS................................................... 170

xi

LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

CID Collision-induced dissociation

Da Dalton

DFT Density function theory

DHB 2,5-dihydroxybenzoic acid

DNA Deoxyribonucleic acid

ECD Electron capture dissociation

EDD Electron detachment dissociation

EPR Electron paramagnetic resonance

ESI Electrospray ionization

ET Electron transfer

ETD Electron transfer dissociation

FELIX Free electron laser for infrared experiments

FT-ICR Fourier transform ion cyclotron resonance

GRE Glycyl radical enzymes

HAT Hydrogen atom transfer

ICR Ion cyclotron resonance

IMR Ion-molecule reactions

xv

IMR-MS Ion-molecule reactions mass spectrometry

IR Infrared

IRC Intrinsic reaction coordinate

IRMPD Infrared multiple photon dissociation

LQIT Linear quadrupole ion trap

m/z Mass-to-charge ratio

MS Mass spectrometry

MS/MS Tandem mass spectrometry

PCET Proton-coupled electron transfer

PFL-AE Pyruvate formate-lyase-activating enzyme

RNA Ribonucleic acid

RNR Ribonucleotide reductases

ROS Reactive oxygen species

SAM S‑adenosylmethionine

SID Surface-induced dissociation

SORI-CID Sustained off-resonance irradiation collision-induced dissociation

SWIFT Stored waveform inverse Fourier transform

UV Ultraviolet

WOC Water-oxidizing complex

1. INTRODUCTION

Till the late 50's a crucial role of free radicals in biological systems was almost

unknown. Before Gerschman et al. discovered free radical influence in oxygen toxicity

[1] and McCord and Fridovich discovered the superoxide dismutase enzymes [2], free

radicals were studied mostly in a context of radiation, polymer and combustion

technology [3].

There are two common ways for radical formation: reactions involved homolytic

bond cleavage, where each part of the molecule ends up with an unpaired electron, and

electron transfer (metal-ion or enzyme-mediated oxidation/reduction of peroxides), or a

combination of both [4]. Odd-electron species are unstable and therefore very reactive,

because of the presence of an unpaired electron. During the interaction with other

molecules, free radicals often cause multiple structural and functional changes [5]. The

main reactions that radicals can undergo are: electron transfer, hydrogen atom

abstraction, addition, and radical substitution. The destructive nature of free radicals is

obvious, although this fact could be both positive and negative. On the positive side they

play an important role in cell metabolism and signaling processes [6], immune protective

mechanisms [7] and enzymatic pathways [4, 8]. At the same time free radicals can

negatively affect important cell molecules (lipids, proteins, carbohydrates, DNA, etc.)

2

[9]. Within a protein, radicals might react via side-chain attack, backbone attack, cross-

linking, causing backbone fragmentation, formation of new radicals, dimerization, etc.

[10]. These reactions can lead to cleavage of the polypeptide chain and to formation of

cross-linked protein aggregates via oxidation, causing loss of structural and functional

activity [11]. Free radicals can attach to unsaturated and polyunsaturated fatty acids

forming new destructive radicals like hydroperoxy radicals and lipid hypoperoxides,

causing lipid peroxidation [12, 13]. Considering the presence of lipids in cell membranes,

this process can potentially damage the entire cell. In addition, free radicals interfere with

mitochondrial aerobic phosphorylation and inhibit synthesis of ATP leading to

cytoskeleton damage [14]. Another way of radical action often leading to cell death is

DNA damage [15]. Free radicals might cause fragmentation, hence formation of

defective DNA which could not be replicated. This harmfully affects the cell cycle and

could potentially lead to many forms of cancer [16]. Atherosclerosis is also attributed to

free-radical-induced [17] as well as alcohol-induced liver damage [18] and lung damage

by radicals in cigarette smoke. Free radicals may also be involved in Parkinson's [19] and

Alzheimer's diseases [20], as well as schizophrenia [21].

Thus it is critical to study free radical interactions and pathways in order to

distinguish their role and nature and obtain important fundamental information that forms

a basis for understanding of radical-involved processes.

3

Current work is focused on the amino-acid-based free radicals which are involved

in enzymatic pathways. These radicals are normally located either at the α-carbon of

glycine [22] or on a reactive amino acid side chain such as cysteine [23], tyrosine [24], or

tryptophan [25].

Scheme 1.1. Glycyl radical.

Glycyl free radicals have the simplest structure among all enzymatic radicals.

Within an enzyme a glycine-based radical is located on the Cα, has a high stability due to

the captodative resonance between electron-donating and electron-withdrawing groups

[26, 27]. Although during experiments in the liquid phase the HO• attack caused

hydrogen abstraction from methylene group followed by the radical formation (due to the

presence of protonated amine this process was not energetically favorable). Experiments

on trimethylamine and α-aminoisobutyric acid prove the possibility for protonated amine

to be a target for the radical attack with a further formation of nitrogen-centered free

radical [4]. Theoretical calculations made by J.Hioe et al. demonstrated that glycyl Cα,

radical is more stable than Cα radicals located on alanyl, cysteinyl, tyrosyl, phenylalanyl,

4

or prolyl residues, regardless of its confirmation within a dipeptide [28]. Discovery of the

storage function of glycyl radical was essential from the evolutionary point of view, since

before it was postulated that the presence of oxygen atom was critical for this purpose.

Now the question about radical-driven processes within bacteria before oxygen became a

part of the natural environment was solved - the radical is transferred from its stored

position (glycyl radicals in the anaerobic case and tyrosyl radical in the aerobic case) for

further action [29]. The reaction cycle of all known glycyl radical enzymes (GREs) is

thought to be initiated by transfer of the radical to the thiol group of a conserved cysteine

located in the middle of the polypeptide chain of the same subunit, which is close enough

to the glycyl radical site to allow the hydrogen atom transfer (HAT) [22, 30-32].

Glycyl radical enzymes (GREs) are important biocatalysts that enable strict

anaerobes and facultative anaerobic organisms to catalyze difficult reactions under

strictly anoxic conditions [30]. The stable and catalytically essential glycyl radical on

G734

of pyruvate formate lyase is formed via direct, stereospecific abstraction of a

hydrogen atom from pyruvate formate-lyase-activating enzyme (PFL-AE) found in

Escherichia coli and other organisms [33, 34]. Considering that G734

residue is buried 8 Å

from the protein surface, this process has to involve a conformational change [22, 35, 36].

Study of the benzylsuccinate synthase enzyme obtained from Thauera aromatica

demonstrated glycyl radical catalyzes the first step in the anaerobic toluene metabolism

[30, 37]. It involves the radical transfer from the glycyl storage site to the thiol of the

5

conserved cysteine, followed by HAT from the methyl group of bound toluene, leading to

an enzyme-bound benzyl radical, which provides a suitable reactant for adding to a

double bond of fumarate as a cosubstrate, forming a benzylsuccinyl product radical that

re-abstracts the hydrogen atom from the active site cysteine and renders the enzyme back

to the catalytically competent radical state [30, 38, 39]. The enzyme

4-hydroxyphenylacetate decarboxylase, obtained from Clostridium difficile (a spore

forming, strictly anaerobic bacterium that causes gastrointestinal infections in humans) is

another member of GREs family, catalyzes the formation of p-cresol as the main

fermentation product of tyrosine [40, 41]. Cresol is toxic enough to allow an active

suppression of other microbes or even complete elimination like with protozoa and

metazoa [42], and may provide growth advantages in a highly competitive conditions.

The discovery of the coenzyme B12-independent glycyl radical enzyme glycerol

dehydratase from Clostridium butyricum in 2003 was a surprise [43]. Like the

B12-dependent glycerol dehydratases, this system is also capable of dehydrating

1,2-propanediol to propionaldehyde in addition to glycerol dehydration [32, 43]. It has

been shown that an active thiyl radical (C433

) is generated at this site as a result of

hydrogen atom abstraction by the active site glycyl radical (G763

) [32, 44, 45]. The

glycerol dehydratases can be easily distinguished by H/D exchange, because the

hydrogen in the B12 enzyme is not exchangeable, while in the glycyl radical enzymes the

hydrogen is transiently attached to sulfur and can be substituted [46]. In class III

6

ribonucleotide reductase from Escherichia coli, which provides the cell with the four

deoxyribonucleotides used for DNA chain elongation and repair, a protein-free radical is

located on the Gly681

residue of the α-polypeptide [47-49]. This radical triggers the first

step of the reduction process abstracting of the sugar H3' atom of the ribonucleotide. The

substrate radical is then reduced to the deoxy form and the glycine residue converts back

to its radical form for the next turnover [49-51]. Other work on class III ribonucleotide

reductase from bacteriophage T4 found the radical to be located on Gly580

and this

position was stated to be an absolute requirement for radical retention and enzyme

activity [52]. One of the recently described enzymes, choline trimethylamine lyase, has a

very different function compared to all aforementioned GREs. The class III

ribonucleotide reductase, as judged by the name, is responsible for the nucleotide

reduction, benzylsuccinate synthase for C−C bond formation, pyruvate formate-lyase and

4-hydroxyphenylacetate decarboxylase for C−C bond cleavage, B12-independent glycerol

dehydratase for dehydration, and choline trimethylamine-lyase for C−N bond cleavage

[53-55]. Choline trimethylamine-lyase utilizes hydrogen atom exchange between Gly821

and Cys489

; the orientation of choline in the active site revealed multiple contacts that

could contribute to binding and catalysis with further formation of trimethylamine [55].

7

Scheme 1.2. The radical transfer via hydrogen atom exchange between glycine and cysteine.

Glycyl radicals are oxygen-sensitive and exposure to oxygen often cleaves the

polypeptide chain at the glycyl radical position with consecutive radical deactivation.

Besides a biological role, this phenomenon has an analytical implementation, since the

radical-hosting residue could be determined (e.g. selective reaction with O2 or with

reactive oxygen species (ROS) in general, can pinpoint the location of glycyl radical)

[56].

Scheme 1.3. Tyrosyl radical.

8

Tyrosyl radical is another example of free radicals that participate in enzymatic

activity. According to Prutz, the tyrosine residue is the easiest to oxidize via formation of

the ring radical cation with the rapid deprotonation which gives phenoxyl radical [57].

Extensive overview of the amino acid radicals' role in enzymes has been done by

Westerlund et al. [58]. In particular, redox-active tyrosinate is present in the active sites

of galactose oxidase, the fungal enzyme that catalyzes the oxidation of a broad range of

primary alcohols to aldehydes. The active site is comprised of a copper ion coordinated to

a redox-active amino acid side chain that has been identified as a tyrosylcysteine

(TyrCys) cofactor formed by post-translational cross-linking between a tyrosine (Y272

)

and a cysteine (C228

) residues [59-62]. Glyoxal oxidase formed from Phanerochaete

chrysosporium has a tyrosyl radical on its active site as well [63-65]. Both are members

of the family of radical copper oxidases and use an isolated, monocopper center to carry

out two-electron redox chemistry [66]. Another example is ribonucleotide reductase

(RNR), a ubiquitous enzyme that catalyzes the conversion of RNA to DNA via long-

distance radical transfer, which is initiated by the activation and reduction of molecular

oxygen to generate a stable tyrosyl radical Tyr122

with a very long (days range) lifetime.

Considering functions of the enzyme, this radical formation is one of the most important

proton-coupled electron transfer (PCET) reactions in nature [67, 68]. This enzyme

utilizes many other amino-acid redox cofactors besides tyrosyl in the conversion of

ribonucleotides to deoxyribonucleotides [69]. The class I enzymes employ O2-dependent

9

chemistry at a di-iron center to generate the driving force necessary to oxidize a tyrosine

adjacent to the metal site. Both oxidized tryptophan and tyrosine residues have been

found in the corresponding enzyme from Anacystis nidulans [70]. The compound I state

of cytochrome c peroxidase involves a heme oxyferryl species and a tryptophanyl cation

radical [71], whereas the peroxidase activity of prostaglandin H synthase requires the

participation of a tyrosyl radical [72, 73]. The cytochrome c oxidase and class I

ribonucleotide reductases involve the participation of tyrosine in both electron and proton

transfers. In addition, the catalytic cycles of bovine liver catalase [74] and lioneate diol

synthase [75] have also been suggested to involve tyrosyl radicals. Tyrosine redox

chemistry is involved in energy transduction in photosynthesis and respiration. Studies

performed in the late 1980s showed that photosystem II contains two redox-active

tyrosines, denoted YZ and YD. Their radicals fill the redox potential gap in multistep

charge-hopping reactions involving several cofactors, which is essential for the unique

water-splitting reactions catalyzed by this enzyme. The time of tyrosine oxidation and

proton loss proves the nature of tyrosine PCET to be strongly influenced by the local

dielectric and H-bonding environment (His190

for YZ and His189

for YD) [76]. Located on

the Z subunit Tyr161

acts as a hole mediator between the water-oxidizing complex (WOC)

and the photo-oxidized chlorophyll dimer [77]. D-subunit Tyr160

forms a H-bond

symmetrical to Tyr161

, but its kinetics is much slower (oxidation is on the scale of

milliseconds; reduction (from charge recombination) is on the scale of hours) [78].

10

Another side-chain radicals, the cysteinyl radical, almost exclusively forms a

sulfur-centered radical via hydrogen atom abstraction (Scheme 1.2), which can rapidly

undergo reversible reaction with oxygen forming peroxy radical (RSOO•). In acidic

conditions and presence of another thiol, a disulfide can be formed, and at the biological

pH, reactions of the thiol anion (RS-) predominate [4]. The whole super family of

S‑adenosylmethionine (SAM) enzymes that catalyze the formation of

S-adenosylmethionine by joining methionine and ATP is built around the [4Fe−4S]

clusters, in which three irons are coordinated by cysteinyl residues and the fourth iron has

a non-cysteine ligand [79, 80]. Another important family of enzymes where the presence

of cysteinyl radical is crucial is ribonucleotide reductases (RNRs). Although all classes of

this enzyme are using different cofactors (class I RNR has a diferric–tyrosyl radical, class

II utilizes adenosylcobalamin (Ado-Cbl); class III utilizes S-adenosylmethionine and an

iron–sulfur cluster to generate a glycyl radical; and class IV is proposed to have a Mn

cofactor adjacent to a tyrosyl radical), all of them generate thiyl radical regardless of the

class [81]. Cross-linked Cys-Tyr radical is believed to be present as a cofactor in the

enzyme family of copper-containing oxidases which catalyze the oxidation of a wide

variety of substrates ranging from small molecules to large peptides [82, 83].

Pyruvate formate lyase utilizes Gly734

for radical storage and a pair of two adjacent

cysteinyl residues (Cys418

and Cys419

). The glycyl radical is thought to generate a thiyl

radical at Cys418/419

needed for homolytic substrate cleavage [23].

11

The tryptophanyl radicals play a key role in PCET. The tryptophan residue

presents one of the most interesting cases as there are two types of side chain radicals that

are biologically important – the tryptophan radical cation (Trp+) (Scheme 1.4a), in which

the charge and unpaired spin are both delocalized over the aromatic π-system of the

indole rings, and the neutral tryptophan indolyl radical (TrpN ) (Scheme 1.4b), where the

unpaired spin is formally located on the indole nitrogen. The tryptophan radical cation

serves as an intermediate in electron transfer between the heme group and the protein

(e.g., Trp191

in cytochrome c peroxidase) [84, 85]. DNA photolyase is an enzyme that

catalyzes the light-activated repair of UV-induced DNA damage [86]. Oxidation of the

Trp382

residue is an intermediate step which generates protonated radical cation [87-89]. It

is surrounded mainly by relatively non-polar amino acids and forms a weak H-bond with

Asn378

. At the same time, Trp306

carries neutral indolyl radical and acts as a proton

acceptor [90].

Scheme 1.4. Tryptophan radical cation (a) and tryptophan neutral nitrogen radical (b).

12

The tryptophan indolyl neutral radical can be thought of as tryptophan radical

cation deprotonated at the indole nitrogen. The neutral tryptophan radical often

participates in oxidation of tyrosine into tyrosine phenoxyl radical. This oxidation,

although formally just a hydrogen atom transfer, actually proceeds via a combination of

electron and proton transfers [91-94]. It can also be involved in electron transfer, like

Trp48

in class I ribonucleotide reductase [86]. Its one-electron oxidation first forms an

intermediate which then forms a Tyr122

phenoxyl radical [95]. Interestingly, iron from the

diferryl center oxidizes Trp48

instead of the closer Tyr122

, which would be

thermodynamically more favorable. The Trp48

side-chain indole proton is surrounded by

a highly polar environment, which plays an important role in modulating its redox

potential [67, 69]. In arsenate reductase Trp122

serves a different function. Experiments

from the Gray laboratory identified Trp122

as an effective hole transport in the

electronically excited metal-to-ligand charge transfer [67, 96]. Tryptophan neutral radical

also was found in the versatile peroxidase from Pleurotus eryngii. It is located on the

protein surface at Trp164

and apparently capable of oxidizing large molecular mass

substrates, which, because of their large size, are not able to reach the active heme site

[97]. Chlorite dismutase, another heme enzyme, utilizes neutral tryptophan radical in the

process of chlorite decomposition into Cl- and O2 [98]. Neutral tryptophanyl radical Trp

62

was also found in hen egg lysozyme [99, 100]. Curiously, when it gets replaced with Tyr

(in human and rat lysozymes), the bacteriolytic activity is enhanced 2-4 fold [101].

13

The two tryptophan-based radical species have been studied by various

experimental and theoretical techniques for decades [102, 103]. Multiple EPR and

theoretical studies showed considerable spin delocalization not only for the canonical

π-radical cation, but also for the TrpN radical [97, 104, 105]. The latter species, although

often shown as a σ-radical located at the indole nitrogen, does not display typical

σ-radical reactivity and has high spin density at several indole ring carbon atoms similar

to those in Indole+ according to Walden and Wheeler [106]. Both the radical cation and

the radical can be reduced by typical antioxidants [107].

Free radicals experimental studies are commonly performed using X-ray

crystallography, pulse radiolysis, and EPR. All those methods have their specific

strengths and weaknesses. Though X-ray crystallography can elucidate the protein

structure utilizing diffraction patterns obtained from a protein crystal, it also can modify

the protein's structure or damage the protein crystals with the radiation from the source.

This process of radiation damage generates photo-electrons that can get trapped in protein

moieties, so this kind of experiment must be carefully performed on the protein structures

which are carrying free radicals [108]. The kinetic rates of the radical reactions is another

crucial piece of information; most of the important rate constants were obtained from

pulsed radiolysis and EPR experiments [109-112]. The first uses a beam of accelerated

electrons which initiate a fast reaction within a sample exposed to it; the second utilizes

spin-trapping compounds (e.g. nitrones or nitroso compounds) to react with short-living

14

radicals producing paramagnetic, relatively long-living nitroxide-based radicals, and their

EPR spectra represent specific features of the initial radicals.

Since all enzymes are dynamic systems, the radical migration (within enzyme and

between enzyme and a substrate) occurs all the time. It is a very complex process of vast

biological importance; hence we would like to gain insight into how it takes place.

Considering all the amino acids, there are many possibilities for the radical initiation on

the side chains with the further migration within a protein. Prutz et al. showed sulfur-

based radicals located whether on methionine or cysteine to be important linkers for the

radical migration. Their study of the disulphide radical anion demonstrated its critical

role in the oxidative and reductive radical transfer. Furthermore, they suggested

directions and nature of the radical transfers in solution, thus the radical initiated on

methionine sulfur-nitrogen three electron species can migrate to the tryptophan indole

nitrogen with further formation of the tyrosyl radical, which can be reversibly transferred

to the cysteine sulfur [113]. It has been shown in the example of the class I ribonucleotide

reductase, which consists of two homodimeric subunits, that the initial stable tyrosyl

radical (Y•) on one subunit generated a thiyl radical (S

•) on a second subunit undergoing

the long-range migration of 35 Å. In contrast with that in most biological systems, redox

cofactors are separated by 10-14 Å [114]. Another example of such a transfer happens in

spore photoproduct lyase between Y99

and C141

residues [115]. As was previously

mentioned, radical transfer from glycine to the cysteine thiol group is the main step of

15

GREs catalysis [32]. Important adjacent factors, such as radical transfer distance,

conformational freedom as well as pH, were discovered during these studies (Table 1.1).

The transfer was initiated pulse radiolytically by azide radicals, which react selectively

by electron abstraction from tryptophan [116].

Table 1.1. Rate constants of intramolecular radical reactions of between tryptophan and tyrosine within a

peptide (adopted from [4]).

Substrate Process Rate constant (s-1

)

TyrOH-TrpNH TrpN• → TyrO

• 5.4 - 6.7 × 10

4

TyrOH-Pro-TrpNH TrpN• → TyrO

• 7.2 × 10

3

TyrOH-(Pro)2-TrpNH TrpN• → TyrO

• 3.2 × 10

3

TyrOH-(Pro)3-TrpNH TrpN• → TyrO

• 2.0 × 10

3

TrpNH-TyrOH TrpN• → TyrO

• 7.3 - 7.7 × 10

4

TrpNH-TyrOD (in D2O) TrpN• → TyrO

• 2.3 × 10

4

D-TrpNH-L-TyrOH TrpN• → TyrO

• 1.5 × 10

5

TrpNH-Gly-TyrOH TrpN• → TyrO

• 5.1 × 10

5

TrpNH-(Gly)2-TyrOH TrpN• → TyrO

• 2.4 × 10

4

TrpNH-(Gly)3-TyrOH TrpN• → TyrO

• 3.5 × 10

4

TrpNH-Val-TyrOH TrpN• → TyrO

• 6.6 × 10

4

TrpNH-Pro-TyrOH TrpN• → TyrO

• 2.6 × 10

4

TrpNH-(Pro)2-TyrOH TrpN• → TyrO

• 4.9 × 10

4

TrpNH-(Pro)3-TyrOH TrpN• → TyrO

• 1.5 × 10

3

16

As can be seen from Table 1.1, the radical transfer between tryptophan and

tyrosine varies from multiple factors. Tryptophan-to-tyrosine radical migration involves a

hydrogen atom abstraction process, and when the corresponding hydrogen atom was

substituted with a heavier deuterium atom, the reaction rate was almost three times

reduced (7.3-7.7×104 s

-1 vs. 2.3×10

4 s

-1), due to the kinetic isotopic effect. When two

amino acids are represented as L-isomers, the radical migration will occur more slowly

than when the process involved D-tryptophan and L-tyrosine

(7.3-7.7×104 s

-1 vs. 1.5×10

5 s

-1). This tendency must be due to the orientations more

favorable for the radical transfer of amino acid side chains within a peptide. The spacing

between tryptophan and tyrosine also affects the reaction rate, although there is no direct

correlation with the number of spacers. For example, the radical migration occurred faster

in WGY substrate than in WY (7.3-7.7×104 s

-1 vs. 5.1×10

5 s

-1), but an additional glycine

slowed down the rate by an order of magnitude (5.1×105 s

-1 vs. 2.4×10

4 s

-1). At the same

time migration within WGGGY substrate was faster than WGGY (3.5×104 s

-1 vs.

2.4×104 s

-1). A possible explanation might involve the combination effect of both

distance and conformational freedom change. Extra glycine brings more rotational

freedom in the φ (rotations of the polypeptide backbone around the bonds between N-Cα)

and ψ (rotations of the polypeptide backbone around the bonds between Cα-C) directions

and flexibility to the peptide backbone, giving a more optimal position for the radical

transfer from the tryptophan side chain to the tyrosine side chain.

17

The importance of the substrate conformational freedom could be concluded from

the apparent trend between rigidity of the spacer and the rate of the reaction, therefore the

radical migration will occur the fastest within WGY substrate, comparing to the more

rigid WVY and the most rigid WPY (5.1×105 s

-1 vs. 6.6×10

4 s

-1 vs. 2.6×10

4 s

-1,

respectively). Similar to the glycine spacers, there was no direct trend observed between

the radical migration reaction rate and the number of prolines separating tryptophan and

tyrosine. However compared to the glycine, the more rigid proline spacer slowed down

the reaction by an order of magnitude. While apparently two proline spacers were better

for radical migration process than two glycines (4.9×104 s

-1 vs. 2.4×10

4 s

-1 respectively),

a higher number of glycine spacers enhances the conformational freedom of the peptide,

hence increases the proximity of the corresponding moieties involved in the radical

migration process, while more proline spacers consequently slows down the rate of the

reaction, as seen in the table.

The radical migration direction is another important factor that affects the rate of

the reaction, although the presence of adjacent amino acids contributes to the rate change

as well. For instance, within dipeptides WY and YW the difference between rates is

relatively small (7.3-7.7×104 s

-1 vs. 5.4-6.7×10

4 s

-1), as with WPPPY and YPPPW

(1.5×103 s

-1 vs. 2.0×10

3 s

-1), but comparison of the WPPY and YPPW pair demonstrated

an order of magnitude difference (4.9×104 s

-1 vs. 3.2×10

3 s

-1).

18

The gas-phase experiments provide valuable insight compared to solution phase

as they probe the radical species directly, and reveal the intrinsic properties of the

compound without solvent or counter-ion effects. Since amino acid radical enzymes are

very complex structures which might include multiple protein subunits and cofactors, in

our work we are trying to reduce complexity of the system by studying the smaller-scale

models. The investigation of gas-phase radicals has generated a great interest as a method

of modeling and exploring the nature and reactivity of radicals in biological systems.

Understanding the electron transfer (ET) and proton-coupled electron transfer (PCET),

including the distance, reversibility and pathways of these processes is critical for the

explanation of radical migration [69]. Mass-spectrometry in combination with the

gas-phase ion-molecule reactions (IMR-MS) can form, detect and store the relatively

unstable radical for a time long enough to perform required manipulations. IMR-MS can

monitor product formation at a milliseconds scale and consumes very small quantities of

chemicals. The setup to conduct IMR could be coupled with multiple types of MS

instruments. The concentration of the sample required for the analysis is in the nanomolar

scale or lower, which makes IMR suitable for analysis of rare or expensive samples,

makes it perfect choice for the free radicals investigations. Research in this field provides

important information that forms a basis for understanding processes where radicals are

involved.

One of the most interesting aspects of the radical research is a study of the

19

radical-induced fragmentation, since most of the time experimental results are very

different from the one of their even-electron counterparts. The presence or absence of any

signature fragments in combination with knowing the exact initial location of the radical

(using regiospecific formation methods) could give fairly certain results regarding

radical-driven processes within a peptide, which later could be confirmed with theoretical

calculations, spectroscopy methods, etc. Under a low-energy collision-induced

dissociation (CID) conditions the fragmentations patterns of a protonated, even-electron

peptide are charge-driven, thus the side chains of the amino acid residue often remain

intact, and mostly b and y ions (with common losses of NH3 or H2O) could be observed

[117]. With the presence of an unpaired electron these patterns will change to both

charge- and/or radical-driven. This phenomenon became a basis for alternative tandem

mass spectrometry dissociation techniques involving unpaired electron-induced

fragmentation, such as electron-transfer dissociation (ETD), electron-capture dissociation

(ECD) and its negative-ion mode equivalent – electron-detachment dissociation (EDD).

ECD is a powerful proteomics tool, which is able to produce c and z-ions by cleaving

backbone sites of the multiply charged proteins and preserve post-transitional

modifications, as opposed to existing CID, IRMPD, UV-photodissociation, SID, and

electron-impact dissociation which were producing mostly b and y-ions often cleaving

post-transitional modifications. This fragmentation pathway was explained as a

nonergodic dissociation of radical ions via intramolecular electron transfer to the more

20

stable backbone site before their stabilization [118]. This technique is critical in the "Top-

Down Proteomics" approach [119]. Later Zubarev's group introduced EDD and they

claimed this technique to be more applicable for the mostly acidic biological proteins,

because in order to apply ECD the protein has to be more basic to be multiply positively

charged after ESI [120]. During experiments an anion was bombarded with >10 eV

electrons and produced electron detachment followed by backbone dissociation, which

led to the production of mostly a, c and z-ions. It has been also concluded that the

electron detachment might occur not only from the anionic surface or carboxy-groups,

but also from the rest of polypeptide. During ionization of the polypeptide chain a mobile

positive radical charge (hole) was created, and its transfer is driven by Coulombic force

towards negative charges. Mutual neutralization of the hole with an electron results in

electronic excitation which causes the bond cleavage [121]. ETD was introduced shortly

after ECD and expanded the radical dissociation to the ion-trap MS. The fragmentation in

this technique was still driven by the electron transfer, although instead of free electrons

used in ECD, ETD employs radical anions. The fragmentation patterns were very similar.

It was concluded that anions with sufficiently low electron affinities could function as

electron donors [122].

21

Scheme 1.5. Examples of hydrogen-rich and hydrogen-deficient radicals (adopted from [123]).

In general, the radical precursor ions can be classified as hydrogen-rich or

hydrogen-deficient based on whether the number of hydrogen atoms is greater or lower

than in the nominal even-electron species, demonstrating a difference in fragmentation

behavior (Scheme 1.5). Hydrogen-rich peptides are typically produced by attachment of

an electron to a multiply protonated peptide ion (forming [M+nH](n−1)+•

), the hydrogen-

deficient peptide cation radicals produced by abstraction of a hydrogen atom or a radical

group from a protonated peptide ion or by loss of an electron from the neutral peptide

([M+(n−1)H]n+•

or M+•

for n = 1). Hydrogen-rich and -deficient peptide cation radicals

will differ by their methods of generation, electronic states, and reactivity [123].

Although peptide radical ions fragmentation under a low-energy CID conditions and

22

during ECD experiments are closely-linked, their fragmentation will differ. During ECD

experiments the hydrogen-rich peptides, depending on their amino acid composition, will

produce characteristic neutral losses due to the side-chain cleavage, along with common

neutral losses from C- and N-termini, the peptide backbone will fragment producing c+

and z•+

- ions (Scheme 1.6) [123], while N-Cα bond dissociation of hydrogen-deficient

peptide radicals will lead to the formation of c/z-ions leading to a different scenario, like

[zn−H]•+

and [cn+2H]+ ions in the case of tryptophan-containing peptides [124]. Also, for

the hydrogen-deficient peptide radicals, it is common to lose amino acid side chains, due

to the radical migration (Scheme 1.7), and the fragmentation pathway of these processes

need to be determined [125].

Scheme 1.6. Typical dissociations of hydrogen-rich peptide cation radicals upon ExD (adopted from [123]).

23

Scheme 1.7. Typical dissociations of hydrogen-deficient amino acid radicals via radical migration (adopted

from [123]).

24

Previously, in our group, we assigned main signature CID fragments related to the

presence of a cysteine radical. Besides common amino acid loss of COOH (-45 Da), there

were some special fragments observed, like -H2S (-34 Da), -CH2SH (-47 Da),

-CH2S (-46 Da) [126]. Siu et al. compared dissociation reactions of the tryptophan- and

tyrosine-containing series of tripeptides, and proposed fragmentation mechanisms. They

obtained mostly [zn-H]•+

or [cn+2H]+ ions, formed via N-Cα bond cleavage [124]. Chu's

group described radical-driven Cβ-Cγ bond cleavage of tryptophan-containing peptides

during low-energy CID, using experimental methods and theoretical calculations. They

observed the neutral loss of 116 Da during the CID of a series of tryptophan-containing

peptide radicals. To come up with the possible scenario of the fragmentation they had to

create the experiment with dehydrogenation of the indole nitrogen atom, proving

Cα–centered radical of the tryptophan residue to be a crucial intermediate in the process

of Cβ-Cγ bond cleavage and exclude HAT from the indole nitrogen from the

fragmentation pathway [127]. In another study, they examined the same problem with a

series of C-terminal tryptophan-containing peptides, where tryptophan was separated

from the N-terminus by a various number of glycine linkers, and with a series of

tryptophan-containing tripeptides, where N-terminal glycine and C-terminal tryptophan

were separated by cysteine, serine, leucine, phenylalanine, tyrosine, and glutamine. They

showed that in C-terminal decarboxylated tryptophan residue within a peptide

competition between the formation of [M−CO2−116]+ and [1H-indole]

•+ systems implies

25

the existence of a proton-bound dimer formed between the indole ring and peptide

backbone, via a protonation of the tryptophan Cγ atom. Within the series of N-terminal

tryptophan-containing peptides, they showed direct cleavage of the tryptophan Cβ-Cγ

bond with the presence of C-terminal basic amino acid. Basic arginine was set apart with

a varying number of glycine spacers and in all cases predominant formation of [M−116]+

product ions has been observed, proving that the basic arginine residue tightly sequesters

the proton and allows the charge-remote Cβ-Cγ bond cleavage to prevail over the charge

directed one [128]. Radical-induced tryptophan Cβ-Cγ bond cleavage has been studied in

Julian's group as well. In their recent work they have published some interesting findings.

They formed a radical on the phenyl ring 2-iodobenzoic acid via photo dissociation,

which was non-covalently bound to RGWALG peptide. The complex was formed by

hydrogen bonding of the guanidinium group of the arginine side chain and the carboxylic

acid in benzoic acid. The fragmentation of the peptide during the photo dissociation was

somewhat unexpected. They observed a homolytic Cβ-Cγ bond cleavage of the tryptophan

side chain leaving a primary radical at the Cβ position. This way tryptophan residue was

converted into the alanine, which is not an energetically favorable pathway because it

would generate two very unstable products, an aryl radical and a primary carbon radical.

The same results were observed within tyrosine-containing peptides. The proposed

mechanism was considering intermolecular process of the homolytic aromatic

26

substitution, where the benzoic acid radical adds to the tryptophan side chain at the Cγ

position and breaks one double bond, leaving a radical at position C2 of the indole ring.

Scheme 1.8. Cβ–Cγ bond cleavage of tryptophan side chain by homolytic aromatic substitution reaction

(adopted from [129]).

In this work they illustrate that intermolecular radical addition at aromatic residues in

peptides can lead to unusual side chain Cβ–Cγ bond or Cα–Cβ bond cleavage in the gas

phase [129]. Hopkinson and co-workers investigated the radical-driven fragmentation of

tyrosine- and tryptophan-containing hexapeptides. They observed unusual formation of

[x+H]•+

and [z+H]•+

fragmentation product ions. Using experimental results and DFT

calculations they proposed a pathway and peptide structural features which favor this sort

of fragmentation [130].

Earlier, Siu's group studied fragmentation of the tryptophanyl radicals around Cα

within various di- and tripeptides with glycines and tryptophan residues at different

positions. They observed a fragmentation pathway which was not common for protonated

27

peptides, such as cleavage of the N-Cα bond and proton transfer from the exocyclic

methylene group in the side chain to the N-terminal residue results in formation of the

[zn-H]•+

ion or cleavage of the Cα-C bond of the peptide containing a C-terminal

tryptophan residue and proton transfer from the carboxylic group to the N-terminal

fragment (a carbonyl oxygen atom) results in formation of the [an+H]•+

ion and

elimination of carbon dioxide [131].

It is worth mentioning that all this research was done on the tryptophan π-radical

cations, produced via oxidation of the peptide during CID of its complex with metal and

an auxiliary ligand [131-133]. Since tryptophan has relatively low ionization energy

(≤ 7.5 eV [134]) and can be oxidized with delocalization of the charge and spin over the

ring systems, more recently Julian explored fragmentation of the tryptophan radical

formed via homolytic cleavage of N-nitrosylated tryptophan, using the method first

reported by Gross [135]. During the N-nitrosylation reaction they formed a very labile

N–NO bond that did not require high collision energy for homolytic dissociation and

could potentially form a radical on the tryptophan residue without fragmenting the

remaining ion, making this technique to be very suitable for the radical research. They

explored peptides of varying basicity to determine the effect of proton mobility on the

competition between radical- and charge-directed dissociation. Experimental results

showed that peptides with higher proton mobility exhibited more proton-catalyzed

fragmentation. At the same time, regardless of proton mobility, the loss of tryptophan

28

side-chain as well as loss of CO2 was observed, because the initial radical can produce

the side chain loss directly, while other fragments require migration of the initial radical.

Also peptides with C-terminal tryptophan residues spontaneously lost CO2 from the

C-terminus during the fragmentation of N–NO bond [136].

The role of tyrosine as a proton-coupled electron transfer cofactor is a topic that

has attracted a lot of attention, and accordingly, represents a focus in multiple studies.

Unfortunately, investigation of the tyrosyl radical in the gas phase is challenging.

Although there are robust methods for the tyrosyl radical formation, like utilization of the

ternary metal complexes to oxidize the amino acid [131, 132], or by Julian's approach via

photo dissociation of the C-I bond (with a subsequent radical migration to the phenoxyl

oxygen) by laser irradiation (266 nm) of protonated 3-iodotyrosine in a specially

modified linear ion-trap mass spectrometer [137, 138], the radical tends to dissociate

spontaneously forming glycyl Cα radical with a loss of tyrosine side chain (-106 Da). The

same trend is happening within tyrosine-containing peptides. The loss of -106 Da has

been described as a signature loss [139], which helped us determine the occurrence of

radical migration between tryptophan and tyrosine side chains. Also this specific side

chain loss was utilized by Siu's group to form isomeric [G•GG]

+, [GG

•G]

+, [GGG

•]+

peptide radical cations, by cleaving side chains of tyrosines of YGG, GYG and GGY

peptide-related ions, respectively [140]. Besides formation during radical-induced

fragmentation of more complex amino acids glycyl radical usually participates in the

29

radical transfers as a very stable structure [141, 142]. Cysteine-to-glycine migration

within a short peptide in the gas phase, which models the final step of GREs action has

been studied previously in our group [143].

The ultimate goal of the presented work is to enhance the understanding of the

radical-driven processes. Our study is aimed to reveal fundamental knowledge about

tryptophan-based radicals. It involves distinct methods for regiospecific radical

generation of π tryptophan radical cation and TrpN• radical. Ternary metal complex

dissociation, where a variety of metals (Cu, Cr, Mn, Fe, etc.) can be used in this reaction

[132, 144-146] to get electron transfer from the neutral Trp to the metal, will be used to

form the π-radical cation Trp+. To generate the H3N

+-TrpN radical via homolytic bond

dissociation, we adopted the N-NO bond cleavage described above. The same methods

will be employed for studying Trp-containing peptides. For the reactivity studies, the

ESI-QIT mass spectrometer modified with a home-built IMR setup is used [147]. IRMPD

spectra and DFT calculations were obtained in collaboration with research groups from

York University, Toronto, Canada; University of Amsterdam, Netherlands, and

University of Melbourne, Australia.

2. STRUCTURE AND REACTIVITY OF THE DISTONIC AND

AROMATIC RADICAL CATIONS OF TRYPTOPHAN

2.1 Introduction

Amino acid-based radicals play important roles in enzyme sites by transforming

substrates via radical chemistry [4]. These radicals are normally located either at the

α-carbon of glycine [22] or on a reactive amino acid side chain such as cysteine [23],

tyrosine [24], or tryptophan [25]. The tryptophan residue presents one of the more

interesting cases as there are two types of side chain radicals that are biologically

important – the tryptophan radical cation (Trp+), in which the charge and unpaired spin

are both delocalized over the aromatic π-system of the indole rings, and the neutral

tryptophan indolyl radical (TrpN ), where the unpaired spin is formally located on the

indole nitrogen (Scheme 2.1).

31

Scheme 2.1. Tryptophan radical cation (a) and tryptophan neutral nitrogen radical (b) in proteins.

The tryptophan radical cation (structure (a) in Scheme 2.1) serves as an

intermediate in electron transfer between the heme group and the protein (e.g. in

cytochrome c peroxidase [84] and DNA photolyase [86]). The tryptophan indolyl neutral

radical (Scheme 2.1b) can be thought of as tryptophan radical cation deprotonated at the

indole nitrogen. It can also be involved in electron transfer (e.g. in ribonucleotide

reductase [86]). However, the neutral tryptophan radical often participates in different

radical pathways such as oxidation of tyrosine into tyrosine phenoxyl radical. This

oxidation, although formally just a hydrogen atom transfer, actually proceeds via a

combination of electron and proton transfers [91-94]. Both the radical cation and the

radical can be reduced by typical antioxidants [107].

The two tryptophan-based radical species have been studied by various

experimental and theoretical techniques for decades [102, 103]. Multiple EPR and

theoretical studies showed considerable spin delocalization not only for the canonical

32

π-radical cation, but also for the TrpN radical [97, 104, 105]. The latter species, although

often shown as a σ-radical located at the indole nitrogen, does not display typical

σ-radical reactivity and has high spin density at several indole ring carbon atoms similar

to those in Indole+ according to Walden and Wheeler [106].

With the significant difficulties in analyzing radicals in whole proteins, much can

be inferred from looking at the simpler radical model systems in the gas phase. Such

gas-phase studies of radical ions have recently been gaining momentum [144, 148-152].

Mass spectrometry (MS) has been at the forefront of those studies due to its mass-

selecting capabilities and a number of chemical techniques have been developed for the

generation of radical ion species with specific location of charge and/or radical.

Both radical species of Trp can be generated via gas-phase techniques. The

tryptophan radical cation can be produced via collision-induced dissociation (CID) of a

redox-active metal ternary complex with neutral tryptophan and an auxiliary ligand

(see eq. 1 for example):

[CuII(terpy)(Trp)]

2+ [Cu

I(terpy)]

+ + Trp

+ (1)

A variety of metals (Cu, Cr, Mn, and Fe, to name just a few) can be used in this

reaction pioneered by Siu and co-workers [132, 144-146]. The electron transfer from the

neutral Trp ligand to the metal has been shown to form the π-radical cation Trp+ (ion 1

33

shown in Scheme 2.2) from Trp and several Trp-containing peptides [127, 128, 153-157]

as opposed to another low-energy structure, the α-carbon captodative radical cation

(ion 3) or the β-carbon radical (ion 4).

Scheme 2.2. Low-energy isomers of tryptophan radical cations: π-radical (1), indolyl nitrogen radical (2),

-carbon radical (3) and -carbon radical (4). Energies calculated by DFT at the B3LYP/6-

311++G(d,p) level of theory from this work (1-3) and from ref [153](4).

The TrpN radical is a neutral species in proteins and as such cannot be studied by

MS techniques easily. Joly et al. were able to form TrpN radical in a small peptide by

electron detachment as a distonic radical anion [158]. In the positive ion mode TrpN

radical can be converted to a distonic radical cation by protonation at the N-terminus,

creating the species isomeric to the π-radical cation Trp+ which we will denote as

H3N+-TrpN (ion 2 in Scheme 2.2). This distonic ion can be generated via CID of

N-nitrosylated protonated Trp as shown in eq. 2:

34

X-NO NO

H3N+-TrpNH H3N

+-TrpN-NO H3N

+-TrpN

(2)

This reaction was first utilized by Hao and Gross [135] as well as Knudsen and

Julian [136] for Trp-containing peptides, and here we employ it for generation of

H3N+-TrpN .

With access to both Trp-based biologically relevant radical species, we here

confirm their structure and study their gas-phase reactivities. Infrared multiple photon

dissociation (IRMPD) spectroscopy in conjunction with MS is one of the widely accepted

gas-phase tools for determining ion structures, including those of radical cations of amino

acids [159-163]. Here we use IRMPD spectroscopy and theoretical calculations to

demonstrate the differences in structures and spectral features of 1 and 2. In addition to

IRMPD, we have recently developed an ion-molecule reactivity approach that allowed us

to confirm the structure and monitor the interconversion between isomeric species of

radical ions of Cys derivatives and Cys-containing peptides [161, 162]. In this work we

explore the gas-phase reactivities of Trp+ and H3N

+-TrpN with the goal of finding

reactions “characteristic” of each isomer, as well as advancing our understanding of their

intrinsic reactivities in a protein environment.

35

2.2 Experimental Section

2.2.1 Chemicals and Reagents

All chemicals and reagents were used as received without any further purification.

L-tryptophan, copper (II) nitrate, 2,2';6',2"-terpyridine (terpy), tert-butyl nitrate,

di-tert-butyl nitroxide, benzeneselenol and 1-propanethiol, were all purchased from

Sigma-Aldrich (Milwaukee, WI). Acetonitrile and methanol were purchased from Fisher

(Pittsburg, PA). Water was purified (18 M ) in-house.

2.2.2 Mass Spectrometry: CID and Ion-Molecule Reactions

Mass spectrometry experiments were carried out at Northern Illinois University

using a Bruker Esquire 3000 quadrupole ion trap mass spectrometer (Bruker Daltonics,

Billerica, MA) modified to conduct ion-molecule reactions as described previously

[8, 164]. The N-nitrosylated tryptophan was generated by allowing a 1.5:1 mixture of

tert-butyl nitrite and a 1 mM solution of Trp (in 50/50 methanol/water with 1% acetic

acid) to react for 10 min. at room temperature. The reaction mixture was diluted a

hundredfold using 50/50 methanol/water with 1% acetic acid and introduced into the ESI

source of the mass spectrometer at a flow rate of 4 µL min-1

. The nebulizer gas, needle

voltage, and temperature were adjusted to about 15 psi, 3.4 kV, and 180oC, respectively.

Radical cation H3N+-TrpN (2) was produced by CID using collision energy sufficient to

dissociate the majority of the precursor ions. For the production of π-radical cation Trp+

36

(1), 200 µL of 1mM tryptophan stock solution (in 50/50 methanol/water) was mixed with

100 µL of 1:1 mixture of 1 mM CuSO4 and 2,2';6',2"-terpyridine stock solutions. The

mixture was then diluted with 700 µL of methanol and immediately introduced into the

ESI source of the mass spectrometer at a flow rate of 4 µL min-1

. The nebulizer gas,

needle voltage, and temperature were adjusted to about 18 psi, 3.4 kV, and 200oC. The

π-radical was obtained through the fragmentation of [CuII(terpy)Trp]

2+ ternary complex

using collision-induced dissociation.

The corresponding radical cations were then mass selected at m/z 204 with a

window of 2 m/z and allowed to react with the neutral reagent introduced into the trap via

a pulsed valve (as previously described [147]) or through a leak valve. The pressure of

neutrals delivered via the leak valve was about 1 x 10-7

Torr in the ion trap region as

estimated by fast ion-molecule reactions. The scan delay was varied from 0 to 10000 ms

allowing the acquisition of mass spectra at different reaction time points.

2.2.3 Infrared Multiple Photon Dissociation Spectroscopy

IRMPD spectroscopy studies were carried out at the Free Electron Laser for

Infrared eXperiments (FELIX) facility located at the FOM-Institute for Plasma Physics

Rijnhuizen (The Netherlands) [165]. A custom-built Fourier-transform ion cyclotron

resonance (FTICR) mass spectrometer [49] equipped with an orthogonal Z-spray ESI

source and a hexapole ion guide was used to sample and subsequently isolate the ions

37

generated by ESI. A dc potential switch was applied to the octapole ion guide

downstream of the hexapole ion guide to transfer the ions into the ICR cell. Operating

parameters for ESI were optimized to maximize formation and transfer of ions to the ICR

cell. The H3N+-TrpN radical cation was produced by front end CID of the N-nitrosylated

tryptophan and isolated for IRMPD studies by using a stored waveform inverse Fourier

Transform (SWIFT) pulse. The π-radical cation Trp+ was produced by isolating the

[CuII(terpy)Trp]

2+ ternary complex in the ICR cell using a SWIFT pulse, followed by

collision activation using a SORI pulse and isolation of the π-radical cation Trp+ using a

second SWIFT pulse. Infrared spectra were collected by monitoring the efficiency of

IRMPD as a function of laser wavelength. Infrared spectra were generated by scanning

the IR wavelength between 5.5 and 8.5 µm in 0.02-0.04 µm increments. The product ion

and precursor ion intensities were measured at each step using the excite/detect sequence

of the FT-ICR-MS after irradiation with FELIX [166]. The IRMPD yield was determined

by normalizing the fragment intensity to the total ion intensity, and linearly corrected for

variations in FELIX power over the spectral range investigated.

2.2.4 Theoretical Calculations

Geometry optimizations and harmonic vibrational frequency calculations were

performed using the Gaussian 09 suite of programs [167] and the hybrid UB3LYP

functional. A relatively small basis set (3-21G*) was used to calculate initial geometry

38

optimizations. All minima located with this basis set were re-optimized using the same

functional and the 6-311++G(d,p) basis set. Transition state calculations were performed

using the QST2 function within Gaussian. In most cases, intrinsic reaction coordinate

(IRC) calculations were used to confirm that the transition states linked the correct

minima that represent pre- and post- transformation structures. Vibrational frequency

calculations were performed to determine whether optimized structures were at minima

(no imaginary frequencies) or first-order transition states (one imaginary frequency), for

zero-point-energy corrections to electronic energies (using unscaled harmonic

frequencies) and for prediction of infrared spectra for comparison to experimental

IRMPD spectra. For comparison of DFT spectra to IRMPD spectra, the computed

frequencies were scaled by a factor of 0.976, which is known to be adequate at the

current level of theory [168]. Theoretical and experimental spectra were also normalized

along the y-axis (relative intensity).

The structures of ions 1 and 2 portrayed in Figures 2.5 and 2.6 were obtained by

starting with all possible combinations of structures which are staggered about the Cα-Cβ

and Cβ-Cγ bonds and subjecting them to structure optimizations. For species 1 the lowest

energy structure is considerably lower than the others and this is attributed to a favorable

through-space interaction between the amino group and the π-system of the indole ring.

The rotamers of species 2 shown in Figure 2.6 all have the NH3+- moiety over the

π-system and consequently are closer in energy.

39

2.3 Results and Discussion

2.3.1 Collision-Induced Dissociation

The two isomeric Trp radical cations were accessed by different chemical

approaches. The classical π-radical cation Trp+ was formed via eq. 1 and the distonic

H3N+-TrpN via eq. 2. First, we compared the behavior of these two species under low-

energy CID conditions in the ion trap. The MS/MS spectra of the two isomeric Trp

radical cations along with one for the protonated Trp are shown in Fig. 2.1. The classical

π-radical cation 1 was studied by low-energy CID previously [131, 133], and our results

(Fig. 2.1, top panel) are fully consistent with the reported findings: the only product is the

3-methyleneindolium ion at m/z 130. The CID spectrum of the distonic radical cation 2

(middle panel) is totally different, with the main fragment ions appearing at m/z 131 (an

ion with a formula C9H9N+, potentially the radical cation of 3-methylindole). A smaller

but significant contribution of the ion at m/z 130 is also present, possibly suggesting a

partial conversion from 2 to 1. In order to get a certain explanation about m/z 130 and 131

peaks presence and their potential formation pathway a set of additional experiments will

be carried out. Minor fragments appear at m/z 186 (loss of water), 176 (potentially a loss

of CO), 158 (loss of CO and H2O) and 144 (a loss of combined C2H4O2). Neither of the

radical cations 1 and 2 displays the loss of ammonia, which is the only fragment in the

CID of TrpH+ (bottom panel).

40

Figure 2.1. CID spectra of: tryptophan radical cation formed via CID of [CuII(terpy)(Trp)]

2+ (eq. 1) (top

panel); tryptophan radical cation formed via CID of H3N+-TrpN-NO (eq. 2) (middle panel);

protonated tryptophan (bottom panel).

Radical cations of amino acids can access lower-energy isomeric structures during

CID, if the barrier to such a rearrangement is comparable to the barriers associated with

fragmentation. For instance, we showed that the CID of distonic radical cation of Cys,

(H3N+-CysS ), leads to several products that can only originate from the isomeric

captodative species [126]. The fact that the two isomeric Trp radical cations fragment

41

very differently suggests little or no interconversion between the species under CID

conditions (the possibility of minimal conversion of 2 to 1 can be inferred from the

common m/z 130 fragment). We investigated the rearrangement of 2 into 1

computationally (Fig. 2.2).

Figure 2.2. B3LYP/6-311++G(d,p) -calculated reaction pathways for the rearrangement of 2 into 1. Values

reported are ΔH at 0K and (ΔG at 298K).

The rearrangement would have to formally involve the transfer of a proton from

the N-terminus to the indole N. The direct transfer leads to a relatively strained TS and a

42

calculated barrier of 142 kJ mol-1

. A slightly lower barrier (137 kJ mol-1

) was calculated

for the concerted process, where a proton is transferred from the carboxyl moiety to the

indole nitrogen together with the proton transfer from the N-terminus to the carbonyl

oxygen (Fig. 2.2). The carboxyl-to-indole N hydrogen transfer was proposed by Knudsen

and Julian [136] as the first step in the fragmentation of peptides with a C-terminal TrpN

radical. While a barrier of 137 kJ mol-1

can certainly be overcome in typical low-energy

CID conditions, there must be other, lower-barrier channels available for the dissociation

of 2, thus making the rearrangement to 1 less competitive and be only a minor channel.

No low-energy pathways from 2 to 3 were found. This compares to the barrier of

126 kJ mol-1

for the rearrangement of 1 to 4 and 195 kJ mol-1

for 1 to 3, as was calculated

by us previously [153]. For comparison, the energy for dissociation of 1 into the m/z 130

ion was found to be about 87 kJ mol-1

[153].

2.3.2 Ion-Molecule Reactions

Previously, we used gas-phase ion-molecule reactions to distinguish between

isomers of cysteine-containing radical ions of amino acid derivatives and small peptides

[8, 161, 162]. The sulfur-based distonic radicals (like H3N+-CysS for Cys) were found to

react readily with allyl iodide and dimethyl disulfide. Captodatively-stabilized α-carbon

radicals did not react with those neutrals. This difference in reactivity allowed us not only

to identify the structure of the radical ion, but also to monitor the S-to-α-carbon radical

rearrangement on the time scale of a typical ion-trap experiment.

43

In this work we used the same approach with the goal of finding ion-molecule

reactions that would differentiate between the two isomers of tryptophan radical cation,

1 and 2. Neither of the species was found to react with allyl iodide or dimethyl disulfide.

However, both isomers reacted with benzeneselenol, PheSeH, by hydrogen atom

abstraction (eq.3),

Trp•

+ PhSeH [Trp+H]

+ PhSe• (3)

and with di-tert-butyl nitroxide, (t-Bu)2NO , via charge transfer (eq.4) (see Fig. 2.3-2.4).

Trp•

+ (t-Bu)2NO Trp + [(t-Bu)2NO]

(4)

With both of these neutrals, the classical radical cation 1 reacted substantially

faster than the distonic species 2. As can be seen in the reaction with

di-tert-butyl nitroxide, about half of the π-radical cation 1 has reacted (Fig. 2.4, bottom

panel), while the conversion of the distonic H3N+-TrpN was only about 15% (top panel).

The fact that both isomeric species participate in the charge transfer reaction mimics their

behavior in proteins. In biological systems both Trp radical cation (equivalent of 1) and

Trp indolyl radical (equivalent of 2) are known to participate in electron transfer

processes [84, 86, 106]. It is also logical that in the gas phase 1 reacts faster than 2.

Reactions of 1 are both charge- and radical-driven, as the charge and the radical are

44

delocalized over the same location – the aromatic π face of indole. By contrast, the

“distonic” species 2 has the radical and the charge in different places which would

necessitate a two-step charge transfer leading to neutral tryptophan. One way would

involve transfer of an electron from an electron donor to the indole nitrogen followed by

a proton transfer from the amino group to the indole nitrogen. Alternatively, an electron

transfer from an electron donor (like di-tert-butyl nitroxide) to the amino nitrogen

followed by a hydrogen atom transfer from the amino group to the indole nitrogen could

take place.

While reactions of 1 and 2 with benzeneselenol or di-tert-butyl nitroxide could be

used to differentiate between the isomers based on their rates, a better test was found in

reaction with n-propyl sulfide, n-PrSH. The π-radical 1 was found to react with propyl

sulfide by a hydrogen atom transfer (eq.5):

Trp+ + n-PrSH TrpH

+ + n-PrS (5)

Under the same conditions, the distonic isomer 2 did not react with propyl sulfide,

as shown in Fig. 2.5. This is in agreement with the lack of reactivity of Trp indolyl

radical towards thiols in solution [4, 107]. This reaction allows us to distinguish between

the Trp+ radical cation 1 and H3N

+-TrpN distonic species 2. In the absence of collisional

45

activation (CID), the barrier for rearrangement from 2 to 1 is too high (136.8 kJ mol-1

see

Fig. 2.2) to make radical migration possible in the tryptophan radical cation.

Figure 2.3. Ion-molecule reaction spectra of 1 (top) and 2 (bottom) with benzeneselenol. Neutral

benzeneselenol was delivered via pulsed valve, reaction time 200 ms (top) and 2000 ms (bottom).

(The presence of m/z 202 and 203 peaks is due to the wide isolation window, not IMR).

46

Figure 2.4. Ion-molecule reaction spectra of 1 (top) and 2 (bottom) with di-tert-butyl nitroxide. Neutral

di-tert-butyl nitroxide was delivered via pulsed valve, reaction time 200 ms (top) and 2000 ms

(bottom).

47

Figure 2.5. Ion-molecule reaction spectra of 1 (top) and 2 (bottom) with n-propyl thiol. Neutral n-propyl

thiol was delivered via pulsed valve, reaction time 50 ms (top) and 2000 ms (bottom).

2.3.3. Theoretical Calculations

Tryptophan indolyl radical 2 generated via eq. 2 is supposed to be, at least

initially, a σ nitrogen-centered radical, as it is produced via a homolytic cleavage of the

N-NO bond of N-nitrosotryptophan. However, as described in the section above, this

isomer failed to react with neutrals like allyl iodide, dimethyl disulfide, and propyl

48

sulfide, thereby displaying behavior that is not characteristic of typical σ nitrogen radical.

In order to explain this discrepancy, theoretical calculations were done on the neutral

nitrogen-based indolyl radical serving as a model of the Trp indolyl radical side chain. In

this planar radical (Cs symmetry) the σ radical has A′ symmetry and the σ- radical has A″

symmetry. The results are shown in Fig. 2.6.

Figure 2.6. B3LYP/6-311++G(d,p) -calculated structures, energies, and electron spin densities (bold

number) of indolyl nitrogen radical (left) and indolyl radical (right).

As can be seen from Fig. 2.6, the σ-radical is an electronically excited state higher

in energy by 146 kJ mol-1

than the ground state, the π indole radical. The excited state

49

does display high spin density (0.83) at the nitrogen, but in the ground state the radical is

delocalized over the nitrogen atom (spin density 0.28), C3 (0.64), C4 (0.27), and

C6 (0.18). These results are fully consistent with calculations published by Walden and

Wheeler more than a decade ago with a much smaller basis set [106]. Extending the

calculation to the full tryptophan indolyl radical 2 does not change the spin density

distribution significantly from that calculated for the 2A″ state of the indolyl radical.

Therefore, while a supposedly highly reactive, nitrogen-centered σ radical is formed

initially, it quickly undergoes an electronic rearrangement either via fluorescence or

internal conversion to the more stable delocalized radical, which explains the lack of

gas-phase reactivity of 2 with allyl iodide, dimethyl disulfide, and propyl sulfide.

Comparison of spin densities of 1 and 2 is given in Figure 2.7. While both species

feature the radical delocalized over the indole π system, the main difference in the density

distribution of 1 is a lower spin at the indole N (0.09 vs. 0.27 in 2) and C3 (0.41 vs.

0.57 in 2) as well as a substantial (0.16) spin at C2 (absent in 2).

50

Figure 2.7. B3LYP/6-311++G(d,p) - calculated structures and spin densities of Trp•+

radical 1 and

NH3+TrpN

• nitrogen radical 2.

2.3.4. Infrared Multiple Photon Dissociation

In the recent past infrared multiple photon dissociation (IRMPD) spectroscopy

has emerged as a powerful tool for studying gas-phase structure of radical ions. IRMPD

was used to assign the structure of His+ as a captodative α-carbon radical [159]. More

recently, this technique was used to successfully distinguish between the sulfur- and

α-carbon-based radicals in several cysteine-containing radical ions 161, 162]. The

distinction was made mostly due to an easily-detectable shift in the position of the C=O

vibration due to resonance in the captodative α-carbon-based radical ions.

Here we report for the first time IRMPD spectra of the two isomeric tryptophan

radical cations 1 and 2, which are given in Figures 2.8 and 2.9, respectively, along with

51

computed spectra for the π-radical (1), the N-radical (2), the captodative α-carbon radical

(3), and the β-carbon radical (4). The experimental IRMPD spectrum of the Trp radical

cation produced via CID of Cu2+

ternary complex is given in Fig. 2.8 (bottom panel). It

has four major groups of peaks: 730-750 cm-1

, a broader feature at1100-1200 cm-1

, an

even broader group of peaks at1350-1550 cm-1

, and 1750-1800 cm-1

. The highest-energy

absorption at 1750-1800 cm-1

corresponds to the C=O stretching mode and can be used to

eliminate the captodative structure 3 where this mode should be red-shifted to 1650 cm-1

due to resonance. For both the π-radical 1 and nitrogen radical 2 the position of the

carbonyl stretch matches the experimental spectrum.

The experimental IRMPD spectrum of the Trp radical cation produced via CID of

N-nitrosotryptophan is given in Fig. 2.9 (bottom panel). It has two major groups of peaks

above 1000 cm-1

: a broad feature at 1100-1200 cm-1

, and a strong absorption at

1750-1800 cm-1

. There are also several weaker absorptions, most notable at

1430-1460 cm-1

, 1540-1580 cm-1

and 1600-1650 cm-1

. Again, the position of the carbonyl

stretch at 1750-1800 cm-1

excludes any contribution of the captodative structure 3.

52

Figure 2.8. Comparison of experimental IRMPD (black trace) of tryptophan radical cation formed via

SORI-CID of [CuII(terpy)(Trp)]

2+ (eq. 1) and DFT-calculated (red trace) IR spectra of 1, 2, 3,

and 4.

53

Figure 2.9. Comparison of experimental IRMPD (black trace) of tryptophan radical cation formed via in-source

CID of H3N+-TrpN-NO

+ (eq. 2) and DFT-calculated (red trace) IR spectra of 1, 2, 3, and 4.

It is not easy to find spectral features that would allow to easily distinguish 1 from

2. The carbonyl asymmetric stretch at 1750-1800 cm-1

appears in both experimental

IRMPD spectra (Figures 2.8 and 2.9, bottom) as one of the major peaks. The carbonyl

stretch is one of the typical diagnostic peaks for determining the structure of radical ions

54

by IRMPD, but in this case both isomers 1 and 2 display it in the same IR region. The

broad features appearing at 1100-1200 cm-1

for both 1 and 2 are most likely the C-O

stretch of the carboxyl coupled together with bending O-H motion. The major different

feature between the two experimental spectra is the broad set of peaks at 1350-1550 cm-1

for 1 which is substantially higher in intensity than the two absorptions at

1430-1460 cm-1

, and 1540-1580 cm-1

for 2. However, it is not straightforward to make

assignment for the peaks in this region.

While the theoretical IR spectra do show various subtle differences, the spectral

resolution and signal-to-noise ratio in the experimental IR spectra is relatively low,

mainly due to the low ion counts obtained for the radical cation species. In addition,

IRMPD intensities may be influenced by the multiple-photon excitation process and

therefore not be directly comparable with calculated intensities [169, 170]. As a

consequence, many of the unique features in the theoretical IR spectra of 1 and 2 are not

prominent enough to identify them unambiguously in the IRMPD spectra. For instance,

one of the main differences between 1 and 2 is the amino terminus (NH2- in 1 versus

NH3- in 2). The protonated amino group in 2 is calculated to have a rocking vibration

mode at about 1120 cm-1

and the “umbrella” motion at 1460 cm-1

. However, the former

peak is masked by a broad absorption at 1100-1200 cm-1

for both 1 and 2, while the latter

may be the small peak at about 1450 cm-1

in the experimental spectrum of 2, but occurs

in the range of the broad group of peaks at 1350-1550 cm-1

for 1, thus making the unique

55

NH3- features not particularly useful for characterizing 2. Conversely, the amino group

NH2- in 1 is calculated to have a rocking mode at 1180 cm-1

and a scissors-type motion at

1600 cm-1

. However, the rocking motion again falls within the broad absorption feature at

1100-1200 cm-1

for 1, whereas the scissors band is not seen in the experimental spectrum

of 1.

We should note that while the “umbrella” mode of NH3- in 2 at 1460 cm-1

is

predicted by the DFT calculations to have high intensity, it is known that such NH2/NH3

modes may be particularly susceptible to anharmonic effects, resulting in a much lower

true intensity and a reduced excitation efficiency in multiple-photon absorption

[171, 172]. Our calculations for this “umbrella” mode indicate substantial anharmonicity

(data not shown), most likely due to the strong hydrogen bonding of one of the NH3

hydrogen atoms to the carboxyl oxygen. It was shown before that the predicted amino

hydrogen inversion mode of the neutral Trp was not present in the experimental FELIX

IR spectrum, which was attributed to anharmonic effects [173].

It was also suggested [106] that the C2-C3 stretch can be used as a distinguishing

feature for the indole radical cation versus indolyl nitrogen radical (side chain models for

1 and 2). In 1, this mode is calculated at about 1470 cm-1

, coincident with a strong

absorption for 2. Conversely, in 2 the C2-C3 stretch is calculated to occur at 1320 cm-1

,

but does not appear in the experimental IRMPD spectrum.

56

Rotation of the Cα-Cβ and Cβ-Cγ bonds of 1 and 2 produces several

conformational isomers and their IR spectra have been calculated by DFT. Interestingly,

the rotamers of 1 yield very different spectra. This phenomenon was previously reported

for protonated tryptophan [174]. The lowest energy isomer 1 (Figure 2.10) has a C=O

stretching mode at ~1730cm-1

, but this peak is blue-shifted to 1778, 1767, and 1771 cm-1

for 1a, 1b, and 1c, respectively. The strong peak at 1362 cm-1

corresponding to the

C3-C9 bond stretching of the pyrrole ring in 1 is less intense in 1a and 1b and is

red-shifted to 1340 cm-1

in 1c. Instead, the Cβ-Cγ stretching mode at ~1467 cm-1

observed

in 1a, 1b and 1c is red-shifted to 1478 cm-1

in 1 with very low intensity only. Another

interesting feature is the moderate absorption at 1076 cm-1

in 1, which corresponds to the

C-N stretching of the backbone. This band becomes weaker in 1a and almost nonexistent

in 1b and 1c. Similarly, the moderate-intensity vibration at 1162 cm-1

(N1-C8 stretching

of the pyrrole ring) and 861 cm-1

(NH2 wagging) in 1a are not obvious in the other

isomers. Recalling that the experimental IRMPD spectrum of π-radical cation of

tryptophan shows broad absorption peaks at 1100-1200, 1350-1550, and 1750-1800 cm-1

,

it is suggested that the broad absorption features are due to a mixture of the rotational

isomers of 1. Indeed, the three rotamers all have energies that are comparable to 1 and the

barriers against interconversions are less than 30 kJ mol-1

.

57

Figure 2.10. Comparison of experimental IRMPD (black trace) of tryptophan radical cation formed via

SORI-CID of [CuII(terpy)(Trp)]

2+ (eq. 1) and DFT-calculated (red trace) IR spectra of four

low-energy conformers of 1. Values reported are ΔH at 0K and (ΔG at 298K).

By contrast, the rotational isomers of 2 display comparable IR spectra

(Figure 2.11). The most intense peaks at ~1739 and 1162 cm-1

correspond to the C=O and

C-OH stretching modes, respectively. The peak at ~1460 cm-1

is the umbrella motion of

the NH3 group while the absorption band at ~1370 cm-1

corresponds to the stretching of

58

the Cα-C bond. The minor peak observed at ~1500 cm-1

of 2a and 2b corresponds to the

breathing mode of the benzene ring.

Figure 2.11. Comparison of experimental IRMPD (black trace) of tryptophan radical cation formed via in-

source CID of H3N+-TrpN-NO

+ (eq. 2) and DFT-calculated (red trace) IR spectra of four low-

energy conformers of 2. Values reported are ΔH at 0K and (ΔG at 298K).

59

2.3 Conclusions

Two isomeric forms of tryptophan radical cation were regiospecifically generated

and studied using various MS-based techniques. The canonical π-radical cation 1 was

produced via Siu‟s method and the distonic indolyl radical protonated at the amino

terminus 2 was produced via homolytic cleavage of the N-NO bond of the protonated

N-nitrosylated tryptophan. The two isomers displayed different fragmentation patterns

under low-energy CID conditions. In an earlier study [153] it was shown that the

rearrangement of 1 into a β-carbon radical 4 with a lower barrier of 126 kJ mol-1

was not

observed under CID conditions (as judged by the fragment ions) because the competitive

dissociation channels occur at lower energy. We suspect it to be the case with 2.

Ion-molecule reactions in the gas phase also showed substantial differences

between 1 and 2. The π-radical cation 1 reacted with n-propyl sulfide via hydrogen atom

transfer while the distonic radical cation 2 did not, in agreement with reactivity of Trp

indolyl radical in solution [107]. Both species reacted with benzeneselenol by hydrogen

atom abstraction and with di-tert-butyl nitroxide via electron transfer, but the π-radical

cation was much more reactive toward both neutrals. The lower reactivity of the distonic

nitrogen radical cation 2 was explained by the unpaired electron being extensively

delocalized over the aromatic ring (as calculated by DFT for both 2 and its side chain

model, indole) and the charge being located at a remote site (amino nitrogen).

60

IRMPD spectroscopy in the 1000-1800 cm-1

region was less effective in identifying

specific features that would aid in distinguishing the two isomers. The most intense bands

of the carbonyl stretch at around 1800 cm-1

(C=O stretch was used for differentiating

between S-based distonic and α-carbon captodative radical cations of Cys derivatives

[161, 162]) and C-O stretch at around 1150 cm-1

were present in the experimental spectra

of both 1 and 2. The 1300-1600 cm-1

region displayed significant differences, but was

also shown to be extremely conformation-dependent, and with several low-energy

structures found by DFT for each isomer it was hard to use features in this region for

definitive assignment. Bellina et al. have recently utilized IRMPD in a different IR region

(3300-3600 cm-1

) that allowed them to observe the indole N-H stretch in the N-acetyl

tryptophan amide radical cation related to 1 [163]. It is expected that this stretch would be

missing in 2 making it a more promising distinguishing feature for the two Trp radical

cation isomers. It is also worth mentioning that other spectroscopic techniques should

offer a useful alternative to IR in comparing the tryptophan neutral radical and the radical

cation in bigger systems. In addition to earlier theoretical calculations of various

spectroscopic properties of TrpN and Trp+ [175, 176], recent experimental works by

Joly et al. [158] and Bellina et al. [163] showed that gas-phase photofragmentation of the

neutral radical occurs at a much lower wavelength as compared to the radical cation

( max of 475 nm vs. 580 nm) which should make the two species easily distinguishable in

the visible range.

3. INVESTIGATION OF FRAGMENTATION OF TRYPTOPHAN

NITROGEN RADICAL CATION

3.1 Introduction

The current work is a follow up on the study of tryptophan-based radicals in the

gas phase [177]. We regiospecifically formed two types of tryptophan side chain radicals,

examined and compared their properties, such as reactivities, infrared spectra, structural

parameters, relative energies and fragmentation patterns. Unlike, the π-radical, where

reactions are both charge- and radical-driven, N-indolyl radical did not react with propyl

sulfide. The reason for the lack of the reaction is the different location of the radical and

the charge, which would necessitate a two-step charge transfer leading to neutral

tryptophan. This process would either involve a transfer of an electron from an electron

donor to the indole nitrogen followed by a proton transfer from the amino group to the

indole nitrogen, or an electron transfer from an electron donor to the amino nitrogen

followed by a hydrogen atom transfer from the amino group to the indole nitrogen. This

was in agreement with the lack of reactivity of H3N+-TrpN radical towards thiols in

solution.

62

Understanding the fragmentation mechanisms of peptide radical cations is always

challenging as they can be driven either by the charge or the radical. The fragmentations

of tryptophan radicals (both π- and „σ-nitrogen‟) exhibit completely different neutral

losses (that are also different from those from protonated tryptophan). Although most of

the neutral losses were successfully identified, one of the unanswered questions was what

is the structure of the ion at m/z 131 derived from H3N+-TrpN . The mechanism by which

it is formed would need to be investigated as well. Tryptophan π-radical produced only

one fragment ion (m/z 130) upon CID, and its structure and the mechanism by which it is

formed was described previously [178, 179]. There was also a study on fragmentation of

a tryptophan σ-N radical within peptide system by Julian's group [180], but that work did

not result in the formation of m/z 131 ion, nor did provide any insight into how the

m/z 131 ion might be formed.

In this chapter we add some new findings and revise some of the information

presented in Chapter 2. The ion at m/z 131 was the major product of CID of N-indole

tryptophan radical H3N+-TrpN , as opposed to the anticipated neutral loss or fragment

formation due to the tryptophan Cα-Cβ bond cleavage (see Figure 3.1). The presence of a

product ion at m/z 130, albeit in lower abundance, suggests that there may be some

conversion of H3N+-TrpN into the Trp π-radical cation prior to dissociation.

Alternatively, the m/z 131 ion might undergo a direct loss of a hydrogen atom to form the

m/z 130 ion. Here the structure of the m/z 131 ion has been optimized using DFT

63

calculations and the possible pathway leading to its formation from H3N+-TrpN

is

examined computationally.



All chemicals and reagents used in this experiment underwent no further

purification following their reception. L-tryptophan, L-tryptophan methyl ester, N-acetyl-

L-tryptophanamide, tryptamine, 3-methylindole, 2,2';6',2"-terpyridine (terpy),

di-tert-butyl nitroxide and tert-butyl nitrate were all purchased from Sigma-Aldrich

(Milwaukee, WI). Methanol and D2O were purchased from Fisher (Pittsburg, PA). Water

was purified (18 M ) in-house.

3.2.2 Mass Spectrometry


using a Bruker Esquire 3000 quadrupole ion trap mass spectrometer Bruker Daltonics,


[8, 164]. The N-nitrosylated tryptophan and derivatives were generated by allowing a

1.5:1 mixture of tert-butyl nitrite and a 1 mM solution of Trp (in 50/50 methanol/water

with 1% acetic acid) to react for 10 min. at room temperature. The reaction mixture was

diluted hundredfold using 50/50 methanol/water and introduced into the ESI source of the

mass spectrometer at a flow rate of 4 µL min-1

. The nebulizer gas, needle voltage, and

64

temperature were adjusted to about 15 psi, 3.4 kV, and 180oC, respectively.

Radical cations H3N+-TrpN were produced by CID using collision energy

sufficient to dissociate the majority of the precursor ions. Deuterated form of the radical

was formed using the same procedure, except with D2O/CH3OD as the solvent.

For the production of 3-methylindole π-radical cation, 200 µL of 1mM tryptophan

stock solution (in 50/50 methanol/water) was mixed with 100 µL of 1:1 mixture of 1 mM

CuSO4 and 2,2';6',2"-terpyridine stock solutions. The mixture was then diluted with

700 µL of methanol and immediately introduced into the ESI source of the mass

spectrometer at a flow rate of 4 µL min-1


temperature were adjusted to about 18 psi, 3.4 kV, and 200oC. The radical was obtained

through the fragmentation of [CuII(terpy)M]

2+ ternary complex using collision-induced

dissociation. The corresponding radical cations were then mass selected with a window

of 2 m/z for further analysis.

Geometry optimizations and harmonic vibrational frequency calculations were

performed using the Gaussian 09 suite of programs [167].


3.3.1 Collision-Induced Dissociation

The ion m/z 131 was mass selected with isolation window of 2 m/z. Gradual

amplification of the collision energy showed proportional increase of m/z 130 ion

abundance, while absolute intensity of m/z 131 signal consequently decreased

65

(inset Fig. 3.1), suggesting m/z 130 ion to be part of the fragmentation pathway of

H3N+-TrpN , rather than a signature fragment of tryptophan π-radical present due to

isomerization of these two species, as was suggested previously.

Figure 3.1. Fragmentation spectra of tryptophan NH3+TrpN

• (MS/MS/MS of m/z 131 ion on the inset).

Independently 3-methylindole π-radical has been formed via in source

dissociation of the ternary complex [CuII(terpy)M]

2+. Fragmentation trend was the same,

confirming suggested structure of m/z 131 ion.

66

Scheme 3.1. Possible ways of forming 3-methylindole radical cation from different molecules. (Nitrosylation

of L-tryptophan and reduction of 3-methylindole through metal-ligand ternary complex).

During CID experiment H3N+-TrpN distonic radical transforms into canonical

3-methylindole radical and both of these molecules has their distinct role in biology. Thus

tryptophan radicals are involved in enzymatic pathways, oxidation of neurotoxins and

neurotransmitters, act as antioxidants, etc. [106], while 3-methylindole radical causes

acute pulmonary edema and emphysema [181, 182]. Ion-molecule reaction with

di-tert-butyl nitroxide of both aforementioned m/z 131 ions showed a tendency towards

electron transfer. Although tryptophan H3N+-TrpN fragment demonstrated only partial

product formation (m/z 144) while 3-methylindole radical completely reacted at the same

conditions (Fig. 3.2), assuming simultaneous presence of two ions with m/z 131 during

tryptophan H3N+-TrpN fragmentation, as shown on the Scheme 3.6.

67

Figure 3.2. Ion-molecule reactions of m/z 131 ions left panel H3N+-TrpN fragment, right panel

3-methylindole radical with di-tert-butyl nitroxide. Reaction times a) and b) 5 ms.,

c) and d) 500 ms., e) and f) 650 ms.

a) b)

d)

f)

c)

e)

68

The investigation of m/z 131 ion formation pathway started with an assumption

that two hydrogen atoms has to be transferred to the indole nitrogen and methylene group

after Cα-Cβ bond cleavage of tryptophan in order to form 3-methylindole radical cation.

Therefore a set of additional experiments on the tryptophan derivatives (Scheme 3.2)

were carried out, with blocked potential migrating hydrogens from the C-terminus and

the N-terminus.

Scheme 3.2. Structures of tryptophan derivatives used to examine the proposed hydrogen transfers.

In the case of the N-acetyl-L-tryptophanamide N-terminus is protected with the

acetyl group and hydroxyl group is substituted with an amine so both potential

C-terminus and the N-terminus hydrogen atom migrations are excluded, hence we are not

expecting any ion formation from the Cα-Cβ bond cleavage. This assumption was

confirmed during CID experiment of the corresponding radical cation (Fig. 3.3) where no

peaks at m/z 130-131 were observed.

69

Figure 3.3. Fragmentation spectrum of N-acetyl-L-tryptophanamide. (All potential H-transfers are

blocked – no m/z 130/131 ions formed).

During experiments with of L-tryptophan methyl ester we blocked only the

C-terminal hydrogen atom transfer. Fragmentation spectrum demonstrated formation of

m/z 130 ion instead of m/z 131, meaning formation of 3-methyleneindoleum instead of

3-methylindole radical (Fig. 3.4). After we experimentally eliminated the possibility to

form m/z 131 ion, the starting and end points of the hydrogen atom transfer became

obvious, although the pathway still needs to be determined.

NH2

O

HN

N

CH3

OH+

70

Figure 3.4. Fragmentation spectrum of L-tryptophan methyl ester. (C-terminus H-transfer is blocked –

m/z 130 ion formed instead of m/z131 ion).

In our hypothesis regarding formation of m/z 131 fragment, both hydrogens that

we considered for the migration are exchangeable. In order to confirm this, we carried out

an H/D exchange experiment on D3N+-TrpN

(Scheme 3.3).

OCH3

O

NH3+

N

71

Scheme 3.3. Structure of D3N+-TrpN

•.

For this experiment, L-tryptophan was dissolved in D2O to yield a solution with a

1mg mL-1

concentration which was further nitrosylated. We carried out MS/MS/MS of

the nitrosylated complex in order to determine the number of exchangeable hydrogens in

the fragment-of-interest. The obtained peak distribution of the most abundant fragment

suggested hydrogen atoms scrambling had occurred (Scheme 3.4), otherwise we would

observe the transfer of one deuterium to indole nitrogen and a transfer of another

deuterium atom to the Cβ, which would be represented on the spectra by the absence of

m/z 134 peak with the full transformation of the former m/z 131 ion into m/z 133 ion.

However, after exchange the intensity of former m/z 131 now consists of the

intensity contributions from m/z 132, m/z 133 and m/z 134 ions (Fig. 3.5), and the former

m/z 130 ion after exchange experiment is represented with the traces of m/z 130 ion,

m/z 131 ion and a part of m/z 132 ion. The presence of m/z 131 - 134 ions experimentally

pointing a selective exchange of the ring hydrogens, involving total of 6 hydrogens in the

fragmentation: 3 from N-terminus, 1 from C-terminus, and 2 from the indole ring. This

suggestion is in agreement with Turecek's work on protonated tryptophan, where

according to calculations the rates of hydrogen atom transfer to the C2 and C4 positions

72

were similar across the entire relevant energy range, and three orders of magnitude faster

comparing to migration to C3 position. Their results concluded positions C2, C4, and C7

to be the best hydrogen atom acceptors with HA = 108, 95, and 86 kJ mol-1

, respectively.

Also this work indicated Cα–Cβ bond dissociation is greatly favored over the C3–Cβ bond

cleavage [183].

D3N CH C

CH2

OD

O

N

H

H

Scheme 3.4. Positions of the hydrogens involved in the fragmentation process, determined during H/D

exchange experiment (3 from N-terminus, 1 from C-terminus, and 2 from the indole ring).

73

Figure 3.5. Fragmentation spectrum of D3N

+-TrpN

•. (Spectrum on the inset: the presence of m/z 131 - 134

ions experimentally suggests involvement of 6 hydrogens in the fragmentation: 3 from

N-terminus, 1 from C-terminus, and 2 from the indole ring).

3.3.2. Theoretical Calculations

In order to form 3-methylindole radical the initial N-radical have to loose CO2

and NH=CH2 moieties. The possible fragmentation pathway has been calculated based on

the suggestion that isomerization barrier between two types of tryptophan side-chain

radicals is 136.8 kJ mol-1

, so the intermediate transformation steps (Fig. 3.6) should not

exceed this energy barrier (Scheme 3.5). After the Cα-C bond cleavage followed by a

transfer of one deuterium atom to the indole nitrogen (Scheme 3.6) the remaining

molecule after the Cα-Cβ bond cleavage could form the next possible neutral products:

-NCD3; -NCD2H; -NCDH2 with the relative probabilities of 1:3:1, forming ions with

m/z 132, m/z 133 and m/z 134, respectively. Considering initial relative intensity of the

74

m/z 130 ion to be 15% from the relative intensity of m/z 131, after the Cα-Cβ bond

cleavage the next neutral losses are expected: - NCD4; -NCD3H; -NCD2H2 with the

relative probabilities of 1/15: 8/15:7/15, and ions formation with m/z 130, m/z 131 and

m/z 132, respectively.

Scheme 3.5. Calculated isomerization energy barrier for tryptophan radicals. (The enthalpies (ΔHo0) and

free energies (ΔGo298, in parentheses) are relative to tryptophan nitrogen radical all energies

are in kJ mol-1

).

The deviation between predicted and experimental peak distribution can be

explained by partial fragmentation of the higher mass peaks which will switch the

intensities ratio towards lower masses.

The formation of the 3-methylindole radical was followed by the geometry

change, HAT processes and bond cleavage described on the Figure 3.6. The whole

process is initiated with a HAT from the amine group to the C3 of the indole. After a

series of transition geometry changes through the bond rotations (first Cβ−Cγ then

Cα−Cβ), followed with OH group flipping, the Cα−C bond will dissociate. This process

causes HAT from the C-terminus to the indole nitrogen atom. After another HAT from

the N-terminus to Cα and Cα−Cβ bond dissociation two isomeric m/z 131 ions could be

75

potentially formed (Scheme 3.5), both will transform into 3-methylindoleum ion

(m/z 130) during the next stage of the CID.

Scheme 3.6. Hydrogen atoms transfer within tryptophan nitrogen radical during fragmentation.

(The enthalpies (ΔHo

0) and free energies (ΔGo298, in parentheses) are relative to tryptophan

nitrogen radical all energies are in kJ mol-1

).

76

Figure 3.6. Tryptophan NH3

+TrpN

• radical fragmentation path calculations.

(The enthalpies (ΔHo

0) and free energies (ΔGo

298, in parentheses) are relative to tryptophan

nitrogen radical all energies are in kJ mol-1

).

77

In the case of tryptamine nitrogen radical the Cα-Cβ bond scission was the main

dissociation channel with formation of the most abundant m/z 131 fragment, as well as

N-Cα bond dissociation with a formation of m/z 143 ion (Fig 3.7). Although the

mechanism of its formation has to be different from the proposed one for tryptophan

H3N+-TrpN , since there could be no HAT from the carbonyl group. There was also a

m/z 117 ion as an evidence of the C3-Cβ bond dissociation as a competitive process to

Cα-Cβ bond dissociation (since during CID of m/z 131 no m/z 117 was formed). Taking to

consideration difference in tryptophan and tryptamine radical structures and

fragmentation patterns, we assume that protonation of the indole nitrogen of tryptamine

radical is happening via HAT from the N-terminus during the dissociation of one of the

side-chain bonds. Determination of the fragmentation pathway of the tryptamine is a part

of the future work of this project.

Figure 3.7. Fragmentation spectrum of tryptamine N-radical cation.

NH3

+

N

78

3.4 Conclusions

The current work is another example of the crucial role of the radical's position

for the fragmentation of the molecule in the gas phase. The radical-induced fragmentation

mechanism of tryptophan indolyl radical cation H3N+-TrpN under low-energy collision

dissociation has been discovered and described. The main fragment ion m/z 131 is proven

to be 3-methylindole π-radical and the peak at m/z 130 has been shown to be a product of

its collision-induced transformation to 3-methyleneindolium ion. The mechanism of its

formation was postulated based on experimental results of CID, H/D exchange and IMR,

and confirmed by DFT calculations. It involves 6 hydrogens: 3 from N-terminus, 1 from

C-terminus, and 2 from the indole ring. 3-Methylindole radical cation generated in an

alternative way (CID of a ternary copper complex) fragmented similarly

(m/z 131 m/z 130) and demonstrates the same electron transfer reactivity, thereby

providing an independent support for our proposed mechanism. The result of the study

confirmed the overall hypothesis about the fragmentation pathway of tryptophan nitrogen

radical cation H3N+-TrpN in general and the structure and formation of the main ion

fragment at m/z 131 in particular.

The fragmentation of the tryptamine nitrogen radical has been examined

independently to contrast differences in the radical-induced fragmentation patterns with

the absence of carbonyl moiety. Based on CID data the main dissociation channels are

Cα-Cβ bond scission (with a formation of the most abundant m/z 131 fragment) and N-Cα

79

bond dissociation (with a formation of m/z 143 ion). In addition, the presence of

low-abundance m/z 117 ion peak suggests a presence of the C3-Cβ bond dissociation

channel competitive to Cα-Cβ bond dissociation process. Additional experiments and

theoretical studies must be carried out in order to describe all involved processes an

contrast the results with previous findings on H3N+-TrpN radical.

4. STRUCTURE AND REACTIVITY OF THE DISTONIC AND

AROMATIC RADICAL CATIONS OF TRYPTOPHAN-

CONTAINING PEPTIDES

4.1 Introduction

Protein-based radicals within active sites of enzymes play key roles in biological

transformations [143]. As reactive intermediates they can be present only under special

and limited conditions and their lifetime is extremely short due to the high reactivity,

which makes it hard to examine their properties. Mass spectrometry offers a unique

approach to study radical systems, not only because a single radical (even unstable one)

can be isolated for sufficient time to perform an experiment, but also because

experiments in the gas phase reveal intrinsic kinetics of the chemical reactions and

product formation in a solvent-free environment. Research in this field provides

important information that forms a basis for understanding processes where radicals are

involved.

One of the key aspects of the radical research is a study of the radical-induced

fragmentation, since most of the time radical ions dissociate very differently compared to

their even-electron counterparts [184]. The radical ions can be divided into

81

hydrogen-rich or hydrogen-deficient based on whether the number of hydrogen atoms is

greater or lower than in the nominal even-electron species. Hydrogen-rich peptides are

typically produced by attachment of an electron to a multiply protonated peptide ion

([M+nH](n−1)+•

), while the hydrogen-deficient peptide cation radicals produced by

abstraction of a hydrogen atom or a radical group from a protonated peptide ion or by

loss of an electron from the neutral peptide ([M+(n−1)H]n+•

or M+•

for n = 1). Hydrogen-

rich and -deficient peptide cation radicals will differ by their methods of generation,

electronic states, and reactivity [123]. The fragmentation of the hydrogen-deficient

peptide under a low-energy CID conditions compared to the ECD dissociation of the

hydrogen-rich peptide with the same amino acid sequence is going to have similar

character, but will differ by the type of fragments formed. During ECD experiments the

hydrogen-rich peptides will produce characteristic neutral losses due to the side-chain

cleavage, along with common neutral losses from C and N-termini, the peptide backbone

will fragment producing c+ and z

•+- ions [123]. The dissociation of N-Cα bond of

hydrogen-deficient peptide radicals will lead to the formation of c/z-ions via a different

scenario, like [zn −H]•+

and [cn +2H]+ ions in the case of tryptophan-containing peptides

[124]. Also, for the hydrogen-deficient peptide radicals, it is common to display amino

acid side chain loss due to the radical migration [125]. In contrast to the peptide-based

radicals, low-energy CID of a protonated even-electron peptide ions are charge-driven,

thus the side chains of the amino acid residues often remain intact, and mostly b and

y-ions (with common losses of -NH3 or -H2O) could be observed [117]. With the

presence of an unpaired electron these patterns will change to both charge- and/or

radical-driven. This phenomenon became a base for alternative tandem mass

82

spectrometry dissociation techniques involving unpaired electron-induced fragmentation,

such as electron-transfer dissociation (ETD), electron-capture dissociation (ECD) and its

negative-ion mode equivalent electron-detachment dissociation (EDD). They became

powerful proteomics tools, which are able to produce c and z-ions by cleaving backbone

sites of the multiply charged proteins and preserve post-transitional modifications, as

opposed to existing CID, IRMPD, UV-photodissociation, SID and electron-impact

dissociation which were producing mostly b and y-ions and often cleaving post-

transitional modifications. These techniques are critical in the "Top-Down Proteomics"

approach [119].

The radical-induced tryptophan-containing peptide fragmentation has been

studied previously. Siu et al. compared dissociation reactions of the tryptophan- and

tyrosine-containing series of tripeptides, formed via copper-metal ternary complex

dissociation, and proposed their fragmentation mechanisms. They described N-Cα bond

cleavage and obtained mostly [zn-H]•+

or [cn+2H]+ ions during the fragmentation [124].

Chu's group described radical-driven Cβ-Cγ bond cleavage of tryptophan-containing

peptides during low-energy CID, using experimental methods and theoretical

calculations. They observed the neutral loss of 116 Da (tryptophan indole) during the

CID of a series of tryptophan-containing peptide radicals. To develop a theory which will

explain the fragmentation they had to create an experiment with dehydrogenation of the

indole nitrogen atom, proving that the Cα–centered radical of the tryptophan residue is a

crucial intermediate in the process of Cβ-Cγ bond cleavage and exclude HAT from the

indole nitrogen from the fragmentation pathway [127]. They also examined the same

83

problem with a series of C-terminal tryptophan-containing peptides, where tryptophan

was separated from the N-terminus by a various number of glycine linkers, and with a

series of tryptophan-containing tripeptides, where N-terminal glycine and C-terminal

tryptophan were separated by cysteine, serine, leucine, phenylalanine, tyrosine and

glutamine. They showed that in C-terminal decarboxylated tryptophan residue within a

peptide competition between the formation of [M−CO2−116]+ and [1H-indole]

•+ systems

implies the existence of a proton-bound dimer formed between the indole ring and

peptide backbone, via a protonation of the tryptophan Cγ atom. On the series of

N-terminal tryptophan-containing peptides they showed direct cleavage of the tryptophan

Cβ-Cγ bond with the presence of C-terminal basic amino acid. Arginine and tryptophan

were separated by a different number of glycines and regardless of the length of the

peptide the formation of [M−116]+ product ions was observed, indicating that the basic

arginine residue tightly sequesters the proton and allows the charge-remote Cβ-Cγ bond

cleavage to prevail over the charge-directed one [128]. Earlier, they studied a

fragmentation of the tryptophanyl radicals around Cα within various di- and tripeptides

with glycines and tryptophan residues at different positions. They observed an N-Cα bond

cleavage and proton transfer from the exocyclic methylene group in the side chain to the

N-terminal residue resulting in the formation of the [zn-H]•+

ion or the cleavage of the

Cα-C bond of the peptide containing a C-terminal tryptophan residue. They also noted a

proton transfer from the carboxylic group to the N-terminal fragment (a carbonyl oxygen

atom) resulting in the formation of [an+H]•+

ion and elimination of carbon dioxide [131].

84

Radical-induced tryptophan Cβ-Cγ bond cleavage has been studied by Julian's

group as well. They formed a phenyl radical of 2-iodobenzoic acid via photo dissociation,

which was non-covalently bound to RGWALG peptide. The complex was formed by

hydrogen bonding of the guanidinium group of the arginine side chain and the carboxylic

acid in benzoic acid. During the fragmentation they observed a homolytic Cβ-Cγ bond

cleavage of the tryptophan side chain leaving a primary radical at the Cβ position. This

way tryptophan residue was converted into alanine, which is not an energetically

favorable pathway because it would generate two very unstable products, an aryl radical

and a primary carbon radical. The same results were observed within tyrosine-containing

peptides. The proposed mechanism involved intermolecular homolytic aromatic

substitution, where the benzoic acid radical adds to the tryptophan side chain at the Cγ

position and breaks one double bond, leaving a radical at position C2 of the indole ring.

In this work they illustrated that intermolecular radical addition at aromatic residues in

peptides can lead to unusual side chain Cβ–Cγ bond or Cα–Cβ bond cleavage in the gas

phase [129].

Hopkinson and co-workers during their investigation on the tyrosine- and

tryptophan-containing hexapeptide radicals, generated by CID of [CuII(terpy)(peptide)]

•2+

complexes, observed unusual formation of [x+H]•+

and [z+H]•+

fragmentation product

ions. Using experimental results and DFT calculations they estimated a pathway and

peptide structural features which favors this sort of fragmentation [130].

Noticeably, all the aforementioned studies were made on the tryptophan π-radical

cations produced via oxidation of the peptide during CID of its complex with metal and

85

an auxiliary ligand [131-133, 185]. In a more recent study Julian et al. explored

fragmentation of the tryptophan radical formed via homolytic cleavage of N-NO bond in

N-nitrosylated tryptophan, using the method first reported by Gross [135]. Since

tryptophan has relatively low ionization energy (7.3-7.5 eV [134]) and can be oxidized

with delocalization of the charge and spin over the ring systems, during N-nitrosylation

reaction they formed a very labile N–NO bond that did not require high collisional energy

for homolytic dissociation. They explored peptides of various basicity to determine the

effect of proton mobility on the competition between radical- and charge-directed

dissociation. Experimental results showed peptides with higher proton mobility exhibited

more proton-catalyzed fragmentation. At the same time, regardless of proton mobility the

loss of tryptophan side chain as well as loss of CO2 was observed, because the initial

radical can produce the side chain loss directly, while other fragments require migration

of the initial radical. Also peptides with C-terminal tryptophan residues spontaneously

lost CO2 from the C-terminus during the fragmentation of N–NO bond. This process can

be explained with a position of the radical favorable to abstract hydrogen from the

C-terminus with further loss of CO2 following hydrogen atom transfer [136].

An example of the radical-induced fragmentation difference resulting from the

radical position summarized in Table 4.1. The data represents comparison of tryptophan

N-radical, tryptophan π-radical and glycine Cα-radical to the control sample of protonated

GGW peptide (Scheme 4.1). These data is a summary of results of the original

experiments and replication of the previous work of by Siu et al. [131, 140].

86

Scheme 4.1. Structures of a) protonated GGW, b) nGGW•+

, c) πGGW•+

d) G•GW

+ radicals.

As could be seen from the data the protonated peptide under a low-energy

collision-induced dissociation produces expected b and y-ions along with common amino

acid fragments, such as loss of water molecule (-18 Da), C-terminal loss (-45 Da), and

also some of the tryptophan fragments: loss of ammonia (-17 Da) and

3-methyleneindolium (m/z 130). The fragmentation of G•GW

+ radical (located on

N-terminal glycine Cα) is similar to the control sample, except no 3-methyleneindolium

(m/z 130) was observed, and instead of b-ion a [b-H]+ ion has been formed, pointing the

presence of the radical.

87

Investigation of the tryptophan side chain radicals' fragmentation is even more

interesting. For example the loss of CO2 (-44 Da) could be present in both

N and π-radical fragments when tryptophan is a C-terminal residue, although in Julian's

work this neutral loss was assigned exclusively for N-radical, giving a reason to suggest a

partial isomerization of these two types of radicals. Both radicals formed fragments with

m/z 117, 130, 143, 187, common for radical-driven processes within tryptophan-

containing peptides [131], at the same time π-radical had a trace fragment of the regular

y-ion, while N-radical had a regular b-ion and a radical y1*-ion with m/z 204 which is

isobaric with a tryptophan radical cation. Moreover, comparison of the tryptophan

H3N+-TrpN radical fragmentation with this y1

*-ion fragmentation exhibited different

signal intensities of the same main fragments. This can possibly mean that at least a

portion of the y1*-ion has a structure of the tryptophan Cα radical. This suggestion is

plausible based on the calculations of isomerization energy barriers from the Chapter 2

(e.g. π-radical cation to Cβ = 126 kJ mol-1

or π-radical cation to Cα = 195 kJ mol-1

) which

can be certainly overcome in CID conditions.

The presence or absence of any signature fragments in combination with knowing

the exact initial location of the radical (using regiospecific formation methods) could give

a certain results regarding radical-driven processes within a peptide, which later could be

confirmed with theoretical calculations, spectroscopy methods, etc.

Inspired by previous works and our recent study of the tryptophan aromatic and

distonic radical cation [177], current project was extended to tryptophan-containing

peptides with the aim to obtain some insights about their gas-phase reactivity and

88

possible intramolecular radical transfer, utilizing ion-molecule reactions (IMR) and

collision-induced dissociation (CID) within the ion trap mass spectrometer. It provides

important fundamental information that forms a basis for understanding processes

involving radicals.

In this study we utilized both approaches to form two isomeric tryptophan radicals

within a peptide where tryptophan is the C-terminal residue and explored their

fragmentation and reactivity which will be shown on the example of the AW peptide. The

reactivity of the π and TrpN• AW radicals were compared utilizing ion-molecule reaction

with 1-propanethiol. The two radicals can be distinguished by this reaction, due to the

absence of the reaction with TrpN• and rapid reaction with π-radical cation.

Preliminary data can be analyzed through the prisms of “charge location – radical

reactivity” and “charge location – radical fragmentation” relationships. It not only

demonstrates an easy way to distinguish the two tryptophan radical cations, but also

exposes new directions for the future work on electron transfer between amino acids side

chains in larger peptide systems.

Scheme 4.2. Structures of a) πAW•+

, b) nAW•+

radicals.

89 T

able

4.1

. C

om

par

iso

n a

nd

con

tras

t o

f C

ID f

rag

men

ts o

f pro

ton

ated

GG

W,

nG

GW

•+,

πG

GW

•+ a

nd

G• G

W+ r

adic

als.

(m

ino

r fr

ag

men

ts

**

***

***

*a

re i

n i

tali

c) *

- C

ID s

pec

tru

m A

pp

end

ix C

.

G• G

W+ S

tru

ctu

re

b2-H

y1-N

H3

y1

G• G

W+

Lo

ss

Fra

gm

ent

11

4

18

8

20

5

31

8

πG

GW

•+ S

tru

ctu

re

1H

-in

do

le r

adic

al

3-m

et.n

do

liu

m

C9H

10N

+

Fig

. 1

0b

[1

31

]

[z1-H

] •+

y1

M-C

O2

M-H

2O

πGGW

•+

Lo

ss

44

18

Fra

gm

ent

11

7

13

0

13

2

14

3

18

7

20

5

27

4

30

0

31

8

nG

GW

•+ Str

uct

ure

b2

1H

-in

do

le r

adic

al

3-m

et.n

do

liu

m

C9H

10N

+

Fig

. 1

0b

[1

31

]

[z1-H

] •+

y1*

nG

W•+

M-C

O2

M-H

2O

nG

GW

•+

Lo

ss

44

18

Fra

gm

ent

11

5

11

7

13

0

13

2

14

3

18

7

20

4

26

1

27

4

28

3

30

0

31

8

[GG

W+

H]+

Str

uct

ure

b2

3-m

et.n

do

liu

m

C9H

10N

+

y1-N

H3

y1

M-C

OO

H

M-H

2O

[GG

W+

H]+

Lo

ss

45

18

Fra

gm

ent

11

5

13

0

13

2

18

8

20

5

27

3

28

3

30

1

31

9

90




AW, WA, GW, WG, and GGW were purchased from Selleckchem (Houston, TX),

D3-Ala WA* was provided by Dr. Hopkinson York University (Toronto, ON, Canada).

Copper (II) sulfate, 2,2';6',2"-terpyridine (terpy), tert-butyl nitrate, and 1-propanethiol,

were all purchased from Sigma-Aldrich (Milwaukee, WI). Acetonitrile and methanol

were purchased from Fisher (Pittsburg, PA). Water was purified (18 M ) in-house.

4.2.2 Mass Spectrometry: CID and Ion-Molecule Rreactions

Mass spectrometry experiments were carried out on a Bruker Esquire 3000

quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) modified to

conduct ion-molecule reactions as described previously [8, 164]. The N-nitrosylated

tryptophan within a peptide was generated by allowing a 1.5:1 mixture of tert-butyl

nitrite and a 1 mM solution of the corresponding peptide (in 50/50 methanol/water) to

react for 10 min. at room temperature. The reaction mixture was diluted hundredfold with

methanol and introduced into the ESI source of the mass spectrometer at a flow rate of

4 µL min-1

. The nebulizer gas, needle voltage, and temperature were adjusted to about

15 psi, 3.4 kV, and 200oC, respectively. The peptide N-radical was produced by CID of

N-nitrosylated molecule using collision energy sufficient to dissociate the majority of the

precursor ions. For the production of π-radical cation, 200 µL of 1 mM corresponding

91

peptide stock solution (in 50/50 methanol/water) was mixed with 100 µL of 1:1 mixture

of 1 mM CuSO4 and 2,2';6',2"-terpyridine stock solutions. The mixture was then diluted

with 700 µL of methanol and immediately introduced into the ESI source of the mass

spectrometer at a flow rate of 4 µL min-1


temperature were adjusted to about 18 psi, 3.4 kV, and 200oC. The π-radical was obtained

through the fragmentation of [CuII(terpy)M]

2+ ternary complex using collision-induced

dissociation.

The corresponding radical cations were then mass selected with a window of

2 m/z and allowed to react with the neutral reagent introduced into the trap via a pulsed

valve (as previously described [147]).

4.2.3 Infrared Multiple Photon Dissociation Spectroscopy

IRMPD spectroscopy studies were carried out at the Free Electron Laser for

Infrared eXperiments (FELIX) facility located at the FOM-Institute for Plasma Physics

Rijnhuizen (The Netherlands) [165]. A custom-built Fourier-transform ion cyclotron

resonance (FTICR) mass spectrometer [49] equipped with an orthogonal Z-spray ESI

source and a hexapole ion guide was used to sample and subsequently isolate the ions

generated by ESI. A DC potential switch was applied to the octapole ion guide

downstream of the hexapole ion guide to transfer the ions into the ICR cell. Operating

parameters for ESI were optimized to maximize formation and transfer of ions to the ICR

cell. The peptide N-radical cation was produced by front end CID of the N-nitrosylated

tryptophan within a peptide and isolated for IRMPD studies by using a stored waveform

92

inverse Fourier Transform (SWIFT) pulse. Infrared spectra were collected by monitoring

the efficiency of IRMPD as a function of laser wavelength. Infrared spectra were

generated by scanning the IR wavelength between 5.5 and 8.5 µm in 0.02-0.04 µm

increments. The product ion and precursor ion intensities were measured at each step

using the excite/detect sequence of the FT-ICR-MS after irradiation with FELIX [166].

The IRMPD yield was determined by normalizing the fragment intensity to the total ion

intensity, and linearly corrected for variations in FELIX power over the spectral range

investigated.


4.3.1 Collision-Induced Dissociation and Ion-Molecule Reactions

Two types of the tryptophan side chain radicals within a simple dipeptides were

regiospecifically formed the same way as radicals of the single tryptophan. Unlike Siu

and coworkers, who successfully formed radical cation on WGG peptide through the CID

of its Cu2+

ternary complex [131], we were not able to achieve these results on the

peptides where tryptophan was the N-terminal residue. As in the aforementioned work,

the main product during the fragmentation was [M-NH3]+, but ions with m/z that

correspond to the peptide radical cation were found in negligible quantities even after

intensive tune adjustment, which made their isolation impossible. Dissociation of the

ternary complex is shown on the Fig. 4.1.

93

Figure 4.1. CID spectrum of [CuII(terpy)WA]

2+ complex. No WA

•+ ion at m/z 275 is seen.

(Spectrum on the inset MS3 of the m/z 258 ion).

94

Scheme 4.3. Structures of a) πWA•+

, b) nWA•+

radicals.

Unlike the π-radical (Scheme 4.3a), the nWA•+

(Scheme 4.3b) radical was formed

successfully via homolytic cleavage of N-nitrosylated tryptophan of WA dipeptide. As

could be seen from Fig. 4.2, predominant loss of NH3 is still dominant, but the intact

radical is present in noticeable quantities, enough for further analysis.

During subsequent fragmentation of the nWA•+

radical ion (Fig. 4.3) the main

fragment appeared at m/z 159. This is a1+ ion, a common fragment for tryptophan-

containing peptides (other fragments at m/z 77, 117, 130, 132, 170, 171 were assigned

previously [117]). But the ion with m/z 159 results from the loss of 116 Da, which is,

based on the Siu's work, the signature loss for the Cβ-Cγ bond cleavage of the tryptophan

π-radical cation [128].

95

Figure 4.2. CID spectrum of the N-nitrosylated WA peptide.

Despite the fact that the m/z 159 fragment was observed in the nWG•+

CID

spectrum as well (Appendix C), we performed a set of additional experiments on the

N-radical of the WA* peptide where the side chain of alanine residue is labeled with

deuterium atoms. The results shown on Fig. 4.3 indicate no shift in mass regarding to the

presence of deuterium, strongly suggesting that the m/z 159 ion is the a1+ ion, as was

stated previously.

96

Figure 4.3. CID spectra of nWA•+

radical (top panel) and nWA*•+

radical with deuterium-labeled alanine

side chain (bottom panel). (minor fragments are in italic, shifted peaks highlighted with red).

Due to the lack of the πWA•+

radical CID spectrum, we compared the main

fragments of the nWA•+

with protonated WA (Tab. 4.1). The presence of a1+ ion,

3-methyleneindolium, and the loss of NH3 are common for both, although the C-terminal

neutral losses are different. While protonated WA demonstrates minor loss of 44 Da

(-CO2), nWA•+

has a loss of 45Da (-COOH), and a peak with m/z 214 (-NH3+CO2).

97

Table 4.2. Comparison and contrast of CID fragments of protonated WA and nWA radical. (minor fragments

are in italic) *- CID spectrum Appendix A.

WA

[WA+H]+*

nW•A

+

Fragment Loss Structure Fragment Loss Structure

130

3-methyleneindolium 130

3-methyleneindolium

132

C9H10N+ 132

C9H10N

+

144

159

a1+ 159

a1

+

170

171

Fig. 4 [131]

214 61 M-NH3+CO2

230 45 M-COOH

232 44 M-CO2

259 17 M-NH3 258 17 M-NH3

276

[WA+H]+ 275

nW

•A

+

In the case when tryptophan is not the N-terminal residue, both types of radicals

can be formed. Their CID spectra have different features, so both isomeric radicals could

be distinguished by their fragmentation patterns.

98

Figure 4.4. CID spectrum of nAW

•+ radical. (Spectrum on the inset MS

3 of the m/z 204 ion).

Interestingly, both types of radicals demonstrated a neutral loss of 44 Da (-CO2).

Previously, the same neutral loss was observed in Siu's work on the π-radical and Julian's

work on N-radical, suggesting that the location of the tryptophan at C-terminus is more

important factor than the type of the radical. At the same time protonated AW peptide

(Tab. 4.2) demonstrates a neutral loss of 46 Da (-CO-H2O), suggesting that the loss of

CO2 is a radical-driven process, which might also indicate N-to-π-radical conversion,

since this process includes HAT to the indole nitrogen.

99

Figure 4.5. CID spectrum of πAW•+

radical.

The major peak in both cases was m/z 187 ion, which was previously postulated

by Siu's group to be [z1-H]•+

. Another interesting fragment of nAW•+

is m/z 204 ion, it

was suggested to be a H3N+-TrpN

•, but its CID spectrum (inset Fig. 4.4) was different

from one of the H3N+-TrpN

• (Fig. 2.1). All the fragments were the same, although the

most abundant peaks were different (m/z 186 vs. m/z 131), suggesting the radical

migration, which mechanism needs to be determined in the future work.

100

Figure 4.6. Ion-molecule reaction of πAW•+

with n-propyl thiol. (The product peak on the inset).

101

T

able

4.3

. C

om

par

iso

n a

nd

con

tras

t o

f C

ID f

rag

men

ts o

f pro

ton

ated

WA

an

d n

WA

rad

ical

. (m

ino

r fr

ag

men

ts a

re i

n i

tali

c)

**

***

***

*-

CID

sp

ectr

um

App

end

ix A

.

AW

πA

W•+

Str

uct

ure

3-m

eth

yle

nei

nd

oli

um

a 1+

[z1-H

]•+

y1

M-C

O2

πAW

•+

Lo

ss

44

Fra

gm

ent

13

0

15

9

18

7

20

5

23

1

27

5

nA

W•+

Str

uct

ure

3-m

eth

yle

nei

nd

oli

um

a 1+

[z1-H

]•+

y1

*

M-N

H3+

CO

2

M-C

O2

M-C

O

M-N

H3

nA

W•+

Lo

ss

44

28

17

Fra

gm

ent

13

0

15

9

18

7

20

4

21

4

23

1

24

7

25

8

27

5

[AW

+H

]+*

Str

uct

ure

3-m

eth

yle

nei

nd

oli

um

C9H

10N

+

a 1+

y1-N

H3

y1

M-N

H3+

CO

+H

2O

M-C

O+

H2O

M-H

2O

[AW

+H

]+

Lo

ss

46

18

Fra

gm

ent

13

0

13

2

14

6

15

9

18

8

20

5

21

3

23

0

25

8

27

6

102

Overall, the nAW radical showed a much richer fragmentation, compared to the

π-radical, with the consecutive losses of : NH3, CO (not common), CO2 and NH3+CO2,

while the π-radical had only the loss of CO2, which nevertheless was completely different

from the protonated peptide which tends to lose: H2O, CO+H2O, and NH3+CO+H2O.

4.3.2. Conclusions

Two types of tryptophan side chain radicals were regiospecifically formed within

a short peptide, and their properties were explored via mass spectrometry-based tools.

The canonical πAW•+

radical cation was produced via CID of the ternary metal-ligand

complex, and the distonic indolyl nAW•+

radical was produced via homolytic cleavage of

the N-NO bond of the protonated, N-nitrosylated AW peptide. The two isomers displayed

different fragmentation patterns under low-energy CID conditions.

The same set of experiments were attempted on the WA peptide, but only nWA•+

radical was successfully formed. To eliminate the possibility of the Cβ-Cγ bond cleavage

due to the presence of m/z 159, an additional experiment with nWA*•+

peptide was

performed, where the side chain of alanine residue was labeled with three deuterium

atoms. The results indicated no mass shift in m/z 159, indicating m/z 159 is the a1+ ion, as

was stated previously. Besides a1+ ion, 3-methyleneindolium, and the loss of -NH3,

nWA•+

demonstrated a loss of 45Da (-COOH), and peak with m/z 214 (-NH3+CO2).

Ion-molecule reactions with 1-propanethiol where performed to explore the

reactivity of both radicals. As with the amino acid tryptophan radicals, the π-radical

readily abstracted hydrogen from the thiol while nAW•+

was completely unreactive.

103

These results, besides differentiating the two isomeric species, demonstrated that

reactivity of tryptophan side-chain radicals is not strongly depending on the presence of

an adjacent aliphatic amino acid, and also indicated radical stability and lack of the

N-radical migration from the tryptophan side chain to more stable locations within a short

peptide.

Both radicals were successfully contrasted by their fragmentation patterns and

their reactivity. However, the IRMPD spectroscopy results (Appendix B) were not as

conclusive. The IRMPD experiments were aimed to confirm the lack of the radical

migration from the initial location. Although theoretical spectra had distinct features, the

experimental spectra of both nWA•+

and nAW•+

peptide radicals had very broad bands,

well matching the theoretical spectra of several isomers making definitive conclusions

hard. The structure of the π-radical implies the N-H bond stretch (3460 cm-1

) to be a

signature feature for the radical location elucidation (this band will be absent on the

N-radical spectrum), which was tested previously in Dougurt's group [163]. Also they

contrasted protonated and unprotonated tryptophan along with tryptophan radical in the

UV-VIS region [186]. The characteristic features described in these spectroscopy

experiments can be potentially used for determination of the initial radical location and

for investigation of the radical migration for the future work on this project.

5. GAS-PHASE STUDY OF INTRAMOLECULAR RADICAL

MIGRATION OF TRYPTOPHAN-BASED RADICALS WITHIN

SHORT PEPTIDES

5.1 Introduction

Radical migration is a very complex process of vast biological importance since

many biological processes involve a series of electron or hydrogen atom transfer

reactions between molecules or within a single molecule. In this work, our interest was

concentrated on radicals active within proteins. Considering all the amino acids, there are

many possibilities for the radical initiation on some of the side chains with the subsequent

radical migration within a protein. Prutz et al. showed that sulfur-based radicals located

on methionine or cysteine serves as important linkers for such radical migration. In their

works they suggested directions and the nature of radical transfers in solution. For

instance, the radical initiated on methionine disulfide cation can migrate to the tryptophan

indole nitrogen with the further formation of the tyrosyl radical, which can reversibly be

transferred to the cysteine sulfur [113]. The possible amino acid side chain radical

transfers and interconversions are summarized on Fig. 5.1 [4].

105

Figure 5.1. Amino acid side chain radical interconversion [4].

Since amino acid radical enzymes are very complex structures which often

include multiple protein subunits and cofactors, we set out to examine tryptophan-based

106

radical migration within a smaller scale peptide models in the gas phase at thermal

conditions and with application of additional collision energy. The designed experiments

are aimed to enhance the understanding of two basic radical-driven processes: the

electron transfer (ET) and proton-coupled electron transfer (PCET), and their properties

including the distance, reversibility and pathways. The gas-phase experiments provide

valuable insight compared to the solution phase experiments as they probe the radical

species directly, and reveal the intrinsic properties of the compound without solvent or

counter-ion effects. The current project is aimed to reveal the nature of radical mobility

within short peptides.

5.2 Study of Tryptophan-to-Cysteine Side-Chain Radical Migration

Cysteine along with tyrosine are two amino acids which can be easily oxidized

[4]. Based on the findings from a previous chapter tryptophan π-radical cation in a short

peptide participated in a rapid intermolecular PCET from thiol group. This made us

consider the possibility of the same intramolecular process (Fig. 5.2). Previously, it has

been shown that a radical transfer between tryptophan and methionine disulfide radical

takes place at very acidic pH values in solution [116]. We would like to investigate this

process in the gas phase in solvent-free surround, using all advantages of mass

spectrometry and ion-molecule reactions. For example, collision-induced dissociation of

107

[CuII(terpy)M]

•2+ complex cannot form the radical on cysteine, so the location of the

radical will be on tryptophan side chain [133]. Ion-molecule reaction with such neutral

molecules as allyl iodide or dimethyl disulfide can be utilized to confirm the radical

migration to the cysteine side chain because of their selective reactivity with sulfur-based

radicals [8, 143]. In addition, kinetics (at thermal conditions and with the presence of

external collision energy) of the radical migration can be monitored on the millisecond

scale.

Figure 5.2. Intermolecular formation of S-based radical via PCET of the tryptophan π-radical cation (a),

intramolecular formation of S-based radical via tryptophan-to-cysteine side-chain radical

migration (b).

108

5.2.1 Experimental Section

5.2.1.1 Chemicals and Reagents


CW, CGW, CGGW were purchased from Selleckchem (Houston, TX), copper (II)

nitrate, 2,2';6',2"-terpyridine (terpy), tert-butyl nitrate, dimethyl disulfide were all

purchased from Sigma-Aldrich (Milwaukee, WI). Acetonitrile and methanol were

purchased from Fisher (Pittsburg, PA). Water was purified (18 M ) in-house.

5.2.1.2 Mass Spectrometry: CID and Ion-Molecule Reactions




[8, 164]. The N-nitrosylated tryptophan peptides were generated by allowing a 1.5:1

mixture of tert-butyl nitrite and a 1 mM solution of Trp (in 50/50 methanol/water with

1% acetic acid) to react for 10 min. at room temperature. The reaction mixture was

diluted hundredfold using 50/50 methanol/water with 1% acetic acid and introduced into

the ESI source of the mass spectrometer at a flow rate of 4 µL min-1


needle voltage, and temperature were adjusted to about 15 psi, 3.4 kV, and 180oC,

respectively. Radical cation was produced by CID using collision energy sufficient to

dissociate the majority of the precursor ions. For the production of tryptophan π-radical

cation within a peptide, 200 µL of 1 mM tryptophan stock solution (in 50/50

109

methanol/water) was mixed with 100 µL of 1:1 mixture of 1 mM CuSO4 and

2,2';6',2"-terpyridine stock solutions. The mixture was then diluted with 700 µL of

methanol and immediately introduced into the ESI source of the mass spectrometer at a

flow rate of 4 µL min-1

. The nebulizer gas, needle voltage, and temperature were adjusted

to about 18 psi, 3.4 kV, and 200oC. The peptide radical cation was accessed through the

fragmentation of [CuII(terpy)M]

•2+ ternary complex using collision-induced dissociation

and in-source dissociation.

The corresponding radical cations were then mass-selected with a window of

2 m/z for the further evaluation of their reactivity and fragmentation patterns.

5.2.2 Results and Discussion

5.2.2.1 Collision-Induced Dissociation

In case of the cysteine and tryptophan-containing peptides, dissociation of ternary

metal complex occurring differently comparing to expected scenario (eq.1), producing

neutral radical rather than anticipated radical cation (eq.6), displayed on Fig. 5.3b.

[CuII(terpy)CGW]

•2+ [Cu

I(terpy)]

+ + CGW

• (6)

The spectrum on Fig. 5.3a confirms the complex formation (m/z 331), the

presence of m/z 364 peak on the spectrum has been preliminary assigned as in source

fragmentation of the complex evident by the presence of m/z 296 ion ([CuI(terpy)]

+).

110

With the lack of peptide radicals formed via complex dissociation, ions formed during in

source fragmentation were evaluated.

Figure 5.3. Mass spectra of a) ternary metal complex of CGW, b) CID of [CuII(terpy)CGW]

2+ ion.

CID experiments of the supposed radical cation formed via in-source

fragmentation formed fragments common for the protonated peptide. The most abundant

peak had 9 Da mass difference with precursor ion, along with intense presence of peaks

at the higher mass end, which form a pattern for both evaluated CW and CGW peptides.

111

Figure 5.4. CID spectra of a) m/z 307 ion formed from CW peptide, and b) m/z 364 ion formed from CGW

peptide.

The only possible explanation for obtained CID results is oxidation of cysteine in

solution by copper which leads to the formation of doubly charged S-S dimer. During

CID the dimer tends to lose water molecule (expressed by the most abundant fragment

for both CW and CGW) peptides. Current explanation has been confirmed by the analysis

of the solution where ligand (terpyridine) was excluded from the mixture. Both m/z 307

a)

b)

112

for CW and m/z 364 for CGW were produced and their fragmentation patterns matched

with previously described.

A set of experiments where peptides contain tryptophan N-radical were also

performed, and the fragmentation patterns of nCW•+

, nCGW•+

, and nCGGW•+

were

evaluated. Most likely the radical was initially formed on the tryptophan and no

migration to cysteine occurred, although unlike the π-radical, which can be selectively

formed on tryptophan, in case of N-radical formation both tryptophan and cysteine can

get nitrosylated [8, 177], leaving a place for assumption of complete radical migration

from cysteine-to-tryptophan. To evaluate the initial radical location, the IMR experiment

with dimethyl disulfide has been carried out. This neutral molecule is unreactive towards

tryptophan-based radicals and demonstrated selective reactivity with sulfur-based

radicals. The results of the IMR experiment exhibited formation of the traces of reaction

products (Fig.5.6a), most likely due to the nitrosylation of the cysteine rather than

tryptophan in the fraction of ions. To exclude the scenario with partial radical migration,

additional collision energy has been applied to ions isolated during the IMR experiment

in order to prompt the radical migration if one has taken place. After these manipulations

there was no change in reactivity observed, even after the application of collision energy

sufficient to initiate fragmentation (Fig. 5.6b). As a result of all aforementioned

experiments, we can conclude the initial location of the radical on tryptophan side-chain

and lack of intramolecular tryptophan-to-cysteine radical migration.

113

T

able

5.1

. C

om

par

iso

n o

f C

ID f

rag

men

ts o

f n

CW

•+,

nC

GW

•+,

nC

GG

W•+

rad

ical

s. (

min

or

fra

gm

ents

are

in

ita

lic)

.

nC

GG

W•+

Str

uct

ure

1H

-in

do

le r

adic

al

3-m

eth

yle

nei

nd

oli

um

Fig

. 1

0b

[1

31

]

[z1-H

]•+

y2 -

CO

+H

2O

CG

G•+

y2

M-C

O2

nC

GG

W•+

Lo

ss

44

Fra

gm

ent

11

7

13

0

14

3

17

1

18

5

18

7

21

4

23

5

26

0

37

7

42

1

nC

GW

•+

Str

uct

ure

1H

-in

do

le r

adic

al

3-m

eth

yle

nei

nd

oli

um

Fig

. 1

0b

[1

31

]

[z1-H

]•+

[y1-H

]+

M-C

O2

nC

GW

•+

Lo

ss

44

Fra

gm

ent

11

7

13

0

14

3

16

1

17

8

18

7

20

3

32

0

36

4

nC

W•+

Str

uct

ure

1H

-in

do

le r

adic

al

3-m

eth

yle

nei

nd

oli

um

Fig

. 1

0b

[1

31

]

[z1-H

]•+

M-C

O2

nC

W•+

Lo

ss

44

Fra

gm

ent

11

7

13

0

14

3

18

7

21

3

26

3

30

7

114

Figure 5.5. CID spectra of a) nCW•+

, b) CGW•+

, and c) CGGW•+

.

The results of CID experiments, summarized in the Table 5.1. demonstrated

presence of only tryptophan radical characteristic fragment. The signature neutral losses

115

of cysteine radical: -H2S (34Da), -CH2S (46Da), -CO2,-HS• (77Da) [126] were not

present at all, instead fragments corresponding to Cα-Cβ and Cβ-Cγ radical-induced bond

cleavage of tryptophan along with signature neutral loss of -CO2 (44Da) were observed

[136].

Figure 5.6. Ion-molecule reaction of nCGW•+

with dimethyl disulfide a) no collision energy applied,

b) with collision energy. (reaction time 2000ms).

116

5.3 Study of Tryptophan-to-Tyrosine Side-Chain Radical Migration

Within Short Peptides

According to Prutz the tyrosine residue is the easiest to oxidize via formation of

the ring radical cation with the rapid deprotonation which gives phenoxyl radical

(see Scheme 1.3) [57]. Important factors of the migration from adjacent sides are the

radical transfer distance, conformational freedom of the substrate, and pH. Tryptophan-

to-tyrosine radical migration involves a hydrogen atom abstraction process suggesting

participation of the tryptophan-based neutral N-radical rather than π-radical cation in this

process.

Herein we would like to investigate the possibility of tyrosine oxidation by

regiospecifically-formed tryptophan nitrogen radical and observe the potential radical

migration between their side chains within short peptides.

5.3.1 Experimental Section

5.3.1.1 Chemicals and Reagents


GYW, YGW, and WGY were purchased from Selleckchem (Houston, TX), copper (II)

117

nitrate, 2,2';6',2"-terpyridine (terpy) and tert-butyl nitrate were all purchased from Sigma-

Aldrich (Milwaukee, WI). Acetonitrile and methanol were purchased from Fisher

(Pittsburg, PA). Water was purified (18 M ) in-house.

5.3.1.2 Mass Spectrometry: CID and Ion-Molecule Reactions




[8, 164]. The N-nitrosylated tryptophan within a peptide was generated by allowing a

1.5:1 mixture of tert-butyl nitrite and a 1 mM solution of Trp (in 50/50 methanol/water

with 1% acetic acid) to react for 10 min. at room temperature. The reaction mixture was

diluted hundredfold using 50/50 methanol/water with 1% acetic acid and introduced into



needle voltage, and temperature were adjusted to about 15 psi, 3.4 kV, and 180oC,

respectively. Peptide radical cation was produced by CID using collision energy

sufficient to dissociate the majority of the precursor ions. For the production of π-radical

cations, 200 µL of 1 mM peptide stock solution (in 50/50 methanol/water) was mixed

with 100 µL of 1:1 mixture of 1 mM CuSO4 and 2,2';6',2"-terpyridine stock solutions.

The mixture was then diluted with 700 µL of methanol and immediately introduced into



needle voltage, and temperature were adjusted to about 18 psi, 3.4 kV, and 200oC.

118

The π-radical was obtained through the fragmentation of [CuII(terpy)M]

•2+ ternary

complex using collision-induced dissociation.

The corresponding radical cations were then mass selected with a window of

2 m/z for the further evaluation of their reactivity and fragmentation patterns.

5.3.2 Results and Discussion

5.3.2.1 Collision-Induced Dissociation

Previously, Jovanovic et al. described pH-dependent radical transfer from

tryptophan to tyrosine side chain in solution using pulse radiolysis, via both proton

transfer and electron transfer (Scheme 5.1) [187]. Herein we aimed to examine this

process in solvent-free surroundings using available gas-phase tools. First and foremost,

we are able to regiospecifically form tryptophan nitrogen radical via CID of

N-nitrosylated tryptophan, so we know the initial location of the radical. The obtained

radical did not demonstrate any reactivity towards NO• gas even at longer reaction times

during IMR experiments, which confirmed the initial location as well as proved lack of

the transfer under thermal conditions. CID experiments were performed in order to

supply isolated radicals with additional energy. In this case the occurrence of the radical

migration will be indicated by a signature neutral loss of the tyrosine side chain

(-106 Da). Overall our experiments proved a possibility of the radical migration if the

direction of this process lays towards N-terminus of the peptide. The fragmentation

patterns are shown on the Fig 5.7 and summarized in the Tab 5.2.

119

Scheme 5.1. The mechanism of the radical migration between tryptophan and tyrosine side chains (adopted

from [187]).

Scheme 5.2. Experiments outlining tryptophan-to-tyrosine radical migration investigation via CID.

120

Tab

le 5

.2. C

om

par

iso

n a

nd

con

tras

t o

f C

ID f

rag

men

ts n

GY

W•+

and

nY

GW

•+,

nG

W• Y

+ r

ad

ical

s. (

min

or

fra

gm

ents

are

in

ita

lic)

.

nG

W• Y

+

Str

uct

ure

3-m

eth

yle

nei

nd

oli

um

Ty

r-C

OO

H

y1

a 2

Fig

. 1

e[1

24

]

[b2-H

] •+ (

Trp

)

[a3-H

] •+ (

GG

Y)

c 2

y2-C

O+

H2O

[z2-H

] •+

y2

M-C

O+

H2O

M-N

H3

nG

W• Y

+

Lo

ss

10

3

74

57

46

17

Fra

gm

ent

13

0

13

6

18

2

21

6

22

6

24

3

25

1

26

1

32

1

35

0

36

7

37

8

40

7

42

4

nY

GW

•+

Str

uct

ure

1H

-in

do

le r

adic

al

Ty

r-C

OO

H

Ty

r-N

H3-H

2O

b2-C

O-N

H3

z 1

y1*-H a 2

b2

c 2

M-C

O2-1

16

M-1

06

M-C

O2

M-N

H3

nY

GW

•+

Lo

ss

10

6

56

44

17

Fra

gm

ent

11

7

13

6

14

6

17

6

18

8

20

3

20

5

22

1

23

8

26

4

31

8

36

8

38

0

40

7

42

4

nG

YW

•+

Str

uct

ure

1H

-in

do

le r

adic

al

Ty

r-C

OO

H

Fig

. 1

0b

[1

31

]

[z1-H

] •+

a 2

y1

*(T

rp)

b2

c 2

M-C

O2-1

16

M-1

06

M-C

O2

M-H

2O

nG

YW

•+

Lo

ss

10

6

56

44

18

Fra

gm

ent

11

7

13

6

14

3

18

7

19

3

20

4

22

1

23

8

26

4

31

8

36

8

38

0

40

6

42

4

121

Based on the experimental results some interesting observations can be made. The

signature loss of the tyrosine side chain has been observed in GYW•+

peptide, where two

amino acids of interest, tyrosine and tryptophan, are adjacent to each other and

tryptophan is a C-terminal residue. The presence of the target fragment at m/z 318 is an

evidence of the radical transfer under the low-energy CID conditions. At the same time,

some of the characteristic fragments which can occur only as a result of radical-driven

fragmentation of the tryptophan are present as well, such as m/z 117, 143, 187, 204, 264.

A pair of m/z 117 and m/z 264 came from the signature Cβ-Cγ bond cleavage of

tryptophan radical [128]. We can also conclude that HAT has occurred since protonated

indole was formed (m/z 117). The m/z 187 ion, which was previously postulated by Siu's

group to be [z1-H]•+

, is present as well. The only plausible explanation of this picture is

that two simultaneous independent processes with similar energetics are taking place.

One radical migration occurs within the tryptophan residue from the side chain towards

Cα followed by Cβ-Cγ and Cα-N bond cleavage, the second one between tryptophan and

tyrosine side chains with consecutive HAT from phenol to indole and electron migration

to the tyrosine Cα with further Cα-Cβ bond cleavage and formation of GG•W

+ glycine

Cα-based radical cation. Also a2 and c2-ions (which are not very common in low-energy

CID experiments) were observed during the fragmentation of all three peptides under

investigation.

122

Figure 5.7. Comparison and contrast of CID fragments of a) nGW•Y

+, b) nGYW

•+ and c) nYGW

•+, radical

cations.

Similar results were obtained when glycine spacer was placed between tyrosine

and tryptophan. The main trend remained the same: two simultaneous processes, one is

intratryptophan radical migration; another one is due to tryptophan-to-tyrosine migration.

The absolute abundance of the signature m/z 318 peak is almost the same as in case of

GYW•+

(see Fig.5.7b and c). Although now, based on the abundances of m/z 264 and

[z1-H]•+

ions, Cβ-Cγ bond cleavage is one of the dominant processes. A very abundant

peak of the regular z1-ion was present as well.

123

In the case when tryptophan is no longer a C-terminal residue of the peptide,

radical-driven processes are drastically different (compared to the previous examples).

There was no radical migration between tryptophan and tyrosine observed (intact

tyrosine, an internal fragment, m/z 182 ion was observed instead of the signature m/z 318

ion). In its place, the radical migrated from tryptophan side chain to its Cα, cleaving

tryptophan Cα-Cβ bond with the formation of the pair GG•Y

+ radical ion (m/z 251) and

3-methyleneindolium (m/z 130), the presence of the last one explained by HAT. One of

the possible explanations of this observation is that radicals tend to migrate towards the

N-terminus in this system.

To widen the scope of our investigation, the peptide radicals produced via metal

ion ternary complex dissociation were examined as well. Since it was previously shown

that the radical migration process between tryptophan and tyrosine side chains involves

HAT to the indole nitrogen which is protonated in the π-radical, we did not expect to see

any migration to occur. Another factor of the experiment is the fact that both tryptophan

and tyrosine can be oxidized by the ternary metal complex, so the initial location of the

radical is not certain, although we can assume that during the tryptophan oxidation the

radical will stay intact, while tyrosine oxidation will lead to the observation of the

signature loss of -106 Da during the dissociation of the complex. The spectra shown on

Fig. 5.8 demonstrate that GWY and GYW radical cations (m/z 424) stayed intact, while

dissociation of the ternary complex ([CuII(terpy)YGW]

•2+ at m/z 360) of YGW cations

produced both YGW•+

(m/z 424) and G•GW

+ (m/z 318) radical ions.

124

Figure 5.8. CID of the metal ternary complex with formation of a) GW•Y

+, b) GYW

•+, c) YGW

•+ (all m/z 424)

and G•GW

+ (m/z 318) radicals.

Further analysis of the fragmentation patterns helps make intermediate

conclusions about the processes which are taking place. The main fragmentation pattern

looks similar to that of the N-radicals, described previously regarding to the presence of

the fragments common for the radical site being located on the tryptophan residue

(Fig. 5.9).

125

Figure 5.9. Comparison and contrast of CID fragments of a) GW•Y

+, b) GYW

•+, c) YGW

•+ radicals,

produced via dissociation of copper ternary complexes.

The radical within YGW peptide demonstrated the most interesting results with

regards to the radical transfer between the side chains. Considering immediate formation

of the G•GW

+ (m/z 318) during the ternary metal complex dissociation (Fig. 5.8c) we can

assume that the surviving m/z 424 peak consists of only YGW•+

. Same as the N-radical,

126

the π YGW•+

radical demonstrated evidences of the Cβ-Cγ bond cleavage of tryptophan

radical with formation the pair of m/z 117 and m/z 264 ions. Since originally the charge

on the π-radical is located on the side chain of tryptophan, in order to obtain m/z 264 ion

a charge transfer should take place. Since we postulated that m/z 424 consists of only

YGW•+

and for the radical transfer between the side chains to occur phenoxyl hydrogen

must be abstracted from the tyrosine oxygen, there are two explanations for the presence

of the signature m/z 318 ion. The first one is the m/z 424 consists of the mixture of two

isomeric tryptophan radicals (N and π) and radical transfer occurs only from the

N-radical component of the mixture or N-to-π radical interconversion is taking place

(since the energy barrier of 136 kJ mol-1

[177] can be overcome in typical low-energy

CID conditions). The second scenario includes an initial presence of mixture of stable

tyrosine-based radical (which does not spontaneously lose the side chain) and tryptophan

π-radical, with m/z 318 ion presence due to the initial tyrosine based-radical

fragmentation. Both scenarios are plausible, so additional experiments need to be done in

order to determine the actual mechanism.

127

T

able

5.3

. C

om

par

iso

n a

nd

con

tras

t o

f C

ID f

rag

men

ts o

f G

YW

•+,

YG

W•+

, an

d G

WY

•+ r

ad

ical

s, f

orm

ed v

ia t

ern

ary

co

mp

lex

**

***

***

dis

soci

atio

n.

(min

or

fra

gm

ents

are

in

ita

lic)

.

GW

Y•+

Str

uct

ure

3

-met

hy

lind

ole

Ty

r-C

OO

H

y

1-C

OO

H

y1-N

H3

y1 (

Ty

r)

a 2

Fig

. 1

e [1

24

]

[b

2-H

] •+(T

rp)

M

-CO

2

M

-H2O

GW

Y•+

Lo

ss

7

4

4

4

1

8

Fra

gm

ent

1

31

13

6

1

59

16

5

18

2

2

16

2

26

2

43

3

50

3

80

4

06

42

4

YG

W•+

Str

uct

ure

1H

-in

do

le r

adic

al

[z

1-H

] •+

a 2 b

2 c 2

M-C

O2-1

16

M

-10

6

M

-CO

2

Y

GW

•+

Lo

ss

1

06

6

1

44

Fra

gm

ent

11

7

1

87

2

05

21

5

2

21

2

38

2

64

3

18

3

63

38

0

4

24

GY

W•+

Str

uct

ure

1H

-in

do

le r

adic

al

F

ig.

10

b [

131

]

[z

1-H

] •+

a 2 b

2 c 2

M-C

O2-1

16

M

-CO

2

G

YW

•+

Lo

ss

4

4

Fra

gm

ent

11

7

1

43

1

87

19

3

2

21

2

38

2

64

26

9

3

80

38

8

4

24

128

5.4 Conclusions and Future Work

Current work was aimed to show possible intramolecular radical transfer within

short tryptophan-containing peptides. Peptides which have a N-indolyl radical site on the

tryptophan side chain did not exhibit any migration to cysteine side chain, which was

confirmed by only tryptophan radical-related fragments present in the CID spectra, as

well as IMR results. Although, interestingly, compared to the peptides with no cysteine in

the sequence (CW vs. GW, AW and CGW vs. GGW) cysteine-containing peptides did

not produce m/z 204 tryptophan fragment (Trp•+

), leaving a possibility for the further

investigation. Results of the π-radical migration to the cysteine side chain were not

obtained, since dissociation of the ternary metal complex did not form the peptide radical

cation. Previously, Siu et al. utilized dissociation of copper ternary complexes to form the

radical cation of glutathione [188]. They succeeded with the radical formation and no S-S

bond has been formed during their experiment. Future work needs to be done in order to

optimize the experimental conditions or find appropriate metal-ligand combinations

which dissociation will produce a charged peptide radical.

Based on our experimental results, we believed that radical migration between

tryptophan and tyrosine side chains was observed during low-energy collision-induced

dissociation. This migrations was not seen at the thermal conditions and required external

energy, since all the peptide radicals examined here were formed during more energetic

129

"in-source" fragmentation of the corresponding N-nitrosylated tryptophan-containing

peptides and did not demonstrate a rearrangement before CID experiments (probed by

IMR with NO• gas). In order for the migration to occur, tryptophan has to be closer to the

C-terminus than tyrosine, meaning that radical is migrating towards the N-terminus. In

both cases where tryptophan was a C-terminal residue we observed signature fragments

of both tryptophan and tyrosine radical-driven processes, like the presence of the pair of

m/z 117 and m/z 264 ions, which comes from the Cβ-Cγ bond cleavage or formation of

[z1-H]•+

ion common for tryptophan radical, and the loss of the tyrosine side chain

(-106 Da) as evidence of tyrosyl radical formation. A plausible explanation of this picture

is that two simultaneous independent processes with similar energetics are taking place.

One radical migration occurs within tryptophan residue from the side chain towards Cα

followed by Cβ-Cγ and Cα-N bond cleavage. The second one is between tryptophan and

tyrosine side chains with consecutive HAT from the phenol to the indole moiety with

further tyrosine Cα-Cβ bond cleavage and formation of the glycine Cα-based radical. In

the case when tryptophan was closer to the N-terminus than tyrosine, the radical possibly

migrated from the tryptophan side chain to its Cα, cleaving tryptophan Cα-Cβ bond with

the formation of either GG•Y

+ radical ion (m/z 251) or 3-methyleneindolium (m/z 130).

Surprisingly, considering the necessity of HAT from the tyrosine, we observed an

evidence of the radical migration in πYGW•+

peptide as well. Assuming that m/z 424

consists of only YGW•+

and for the radical transfer between side chains to occur

phenoxyl hydrogen must be abstracted from the oxygen, there are few explanations for

130

the presence of the signature m/z 318 ion. The m/z 424 ion might consist of the mixture of

two isomeric tryptophan radicals (N and π) and radical transfer occurs only from the

N-radical component of the mixture. Also, since the energy barrier of 136 kJ mol-1

can be

overcome in typical low-energy CID conditions, N-to-π radical interconversion could be

taking place. Another possible scenario includes initial presence of a mixture of stable

tyrosine-based radical (which does not spontaneously lose the side chain) and tryptophan

π-radical, with m/z 318 ion presence due to the initial tyrosine based-radical

fragmentation. All aforementioned scenarios are plausible, so additional experiments

need to be done in order to determine the actual mechanism.

6. CONCLUSIONS

The current work was aimed to contribute to the fundamental study of free radical

interactions and pathways to enhance understanding of radical-driven processes. It was

focused on the amino-acid-based free radicals which are involved in enzymatic pathways.

The study of tryptophan-based radicals is especially interesting, because there are two

types of side chain radicals that are biologically important (π-radical cation and N-indolyl

radical), and we can evaluate and contrast their properties.

In order to study tryptophan side chain radicals, we first had to find a way to

model them in the gas phase. With this goal in mind, we followed two different

approaches to regiospecifically form the desired radicals on the single tryptophan amino

acid and to begin study in these simple model systems. The tryptophan π-radical cation

was formed via electron transfer during the gas-phase dissociation of ternary metal

complex ([CuII(terpy)(Trp)]

2+), while an N-indolyl radical was formed by the homolytic

cleavage of the NO group from N-nitrosylated tryptophan during CID. Theoretical

calculations showed that the σ-radical initially formed during the N-nitrosylated

tryptophan dissociation is unstable and rapidly rearranges into the more energetically-

favorable neutral π-radical.

132

The radicals' reactivity has been tested via gas-phase ion-molecule reactions with

benzeneselenol, 1-propanethiol and di-tert-butyl nitroxide. The results of these reactions

can be interpreted from different points of view. First and foremost, due to the charge

presence π-radical cation was found to be more reactive, compared to the distonic

N-indolyl radical cation. It readily reacted with all neutral molecules examined, while the

N-indolyl radical demonstrated only partial reactivity towards benzeneselenol and

di-tert-butyl nitroxide, while it did not react with 1-propanethiol. Selected neutral

molecules can undergo reactions which mimic basic radical-driven processes,

e.g. electron transfer (reaction with di-tert-butyl nitroxide), proton-coupled electron

transfer (reaction with benzeneselenol and 1-propanethiol). IMR 1-propanethiol, besides

its selectivity towards π-radical cation (which can be applied to distinguish two isomeric

tryptophan-based side chain radicals), can imitate the radical interaction with thiol-

containing substrates (cysteine, glutathione, etc.) to some extent. Although active sites of

enzymes are often buried inside the protein with less access to solvent thus closer to

gas-phase conditions, considering IMR as a model tool for intramolecular enzyme

interaction would be inaccurate. Solvent plays a significant role in protein conformation,

bonding, ionization energy, etc., which cannot be addressed via the gas-phase IMR.

However, as was demonstrated in this work, IMR between radicals and

appropriate neutral molecules provides valuable information about potential radical-

induced protein damage as well as some insights about radical-antioxidant interactions.

133

For example, by contrasting reactivity of benzeneselenol and 1-propanethiol we can

assign better antioxidant properties to selenium-containing species compared to the thiol

because it successfully reduced both types of radicals.

Both radicals were then exposed to the low-energy CID in order to elucidate and

contrast their fragmentation patterns. Compared to the protonated, even-electron

tryptophan which tends to lose NH3 during CID, the π-radical formed only one fragment

(3-methylindoleum) during the dissociation of Cα-Cβ bond. Cleavage of the side chain is

commonly considered as an evidence of a radical-driven process. Protonated N-indolyl

radical demonstrated much richer fragmentation which is perhaps both charge- and

radical-driven. In order to estimate its fragmentation pathway, additional experiments

(including H/D exchange, dissociation of tryptophan derivatives and DFT-calculations)

were carried out. The isomerization barrier between the N-indolyl radical and the

π-radical cation was calculated to be 136.8 kJ mol-1

. Since ion-molecule reactions results

postulated the absence of the interconversion between these radicals at thermal

conditions, each transition step in the fragmentation pathway is not supposed to exceed

the isomerization barrier energy. Analysis of the tryptophan derivatives (L-tryptophan

methyl ester, N-acetyl-L-tryptophanamide) fragmentation pointed to the involvement of

N and C-terminal hydrogens in the fragmentation of N-indolyl radical cation. Moreover,

fragmentation analysis of the deuterated N-indolyl radical cation suggested complete

scrambling between the backbone (three from the N-terminus, one from the C-terminus)

and the two aromatic hydrogens (C2 and C4 positions [183]). The experimental results

134

provided conditions for the DFT-calculations and the N-indolyl radical fragmentation

pathway has been postulated. The results of gas-phase IMR di-tert-butyl nitroxide

exhibited similarities in the reactivity of both m/z 131 fragment ion of N-indolyl radical

and independently-formed 3-methylindole radical cation, demonstrating a good

correlation with theoretical calculations.

To confirm our findings regarding the radical position and interconversion,

IRMPD spectroscopy was used as a complimentary technique. Comparison of the

experimental spectra to the computed ones was only partially successful. Because main

features of the experimental spectra were relatively broad, we were able to exclude the

radical migration to Cα, due to the poor match to the theoretical captodative structure

spectrum. However, it was not possible to distinguish the π-radical cation from the

N-indolyl radical in that particular IR region (730 - 1900 cm-1

).

To increase the complexity of the systems studied, both types of radicals were

formed within short di- and tripeptides (AW, WA, GW, WG, and GGW) and contrasted

by their fragmentation and reactivity. The importance of the sequence was confirmed

during the experiments with WA and WG peptides. Since the π-radical cation tended to

lose NH3 from its N-terminus during the radical formation, only N-radicals were

successfully formed. In the case when tryptophan was not the N-terminal residue both

types of radicals were produced, compared and contrasted.

135

As was shown on the example of AW peptide, our findings on the single amino

acid radical cations can be successfully applied to distinguish both types of isomeric

radicals within a peptide as well. Since we have hydrogen-deficient species, their

dissociation pattern was different compared to hydrogen-rich ones. Hydrogen-rich

peptides are typically produced by attachment of an electron to a multiply protonated

peptide ion (forming [M+nH](n−1)+•

), while hydrogen-deficient peptide cation radicals are

produced by abstraction of a hydrogen atom or a radical group from a protonated peptide

ion or by loss of an electron from the neutral peptide ([M+(n−1)H]n+•

or M+•

for n = 1).

The dissociation N-Cα bond of hydrogen-deficient peptide radicals will lead to the

formation c/z-ions via a different of hydrogen-rich species scenario, like [zn−H]•+

and

[cn+2H]+ ions in the case of tryptophan-containing peptides. Also, radical-driven amino

acid side chain loss is common for the hydrogen-deficient peptide radical cations. In

contrast to the peptide-based radicals, low-energy CID of protonated even-electron

peptides are charge-driven, thus the side chains of the amino acid residue often remain

intact, and b and y ions (with common losses of -NH3 or -H2O) could be observed.

During dissociation of both types of radicals within AW peptide radical ions, the

main fragment was formed at the m/z 187, which was previously postulated by Siu's

group to [z1-H]•+

. Another interesting fragment of nAW•+

was m/z 204 ion, which was

suggested to be a H3N+-TrpN

•, but its CID spectrum was different from one of the

H3N+-TrpN

• by the intensities of the most abundant peaks (m/z 186 vs. m/z 131). Overall

nAW radical cation demonstrated much richer fragmentation, compared to the π-radical

136

cation, with the consecutive losses of NH3, CO (not common), CO2 and -NH3+CO2,

while the π-radical cation had only loss of CO2, which, however, was completely

different from the protonated peptide that lost H2O, CO+H2O, and NH3+CO+H2O.

Ion-molecule reactions with 1-propanethiol were performed to explore the

reactivity of both radicals. As with the amino acid tryptophan radicals, the π-radical

cation readily abstracted a hydrogen atom from the thiol while nAW•+

was completely

unreactive. These results, besides providing a method for differentiation of the two

isomeric species, demonstrated no difference in reactivity of tryptophan side-chain

radicals in the presence of an adjacent aliphatic amino acid. This suggests some radical

stability and lack of radical interconversion at thermal conditions, as well as the absence

of migration of the radical site from tryptophan side chain within short peptides.

The IRMPD spectra were obtained for the N-indolyl radical cations for both AW

and WA peptides with the aim to confirm the lack of radical migration from the initial

location. Although calculated theoretical spectra had distinct features, the experimental

spectra of both nWA•+

and nAW•+

peptide radical ions had very broad bands, which

could be matched to most of the theoretical spectra giving no certain structural

information.

A possibility of a radical migration from the tryptophan side chain within peptide

models in the gas phase (at thermal conditions and with application of additional collision

energy) was also examined. The designed experiments were aimed to enhance the

137

understanding of two basic radical-driven processes: the electron transfer and proton-

coupled electron transfer, and their properties including the distance, reversibility, and

pathways. In particular, we attempted to study tryptophan-to-tyrosine-to-cysteine radical

migration system. Based on the previous findings, tryptophan π-radical cation within a

short peptide participated in the very rapid intermolecular proton-coupled electron

transfer from the thiol group, which makes us consider the possibility of the same

intramolecular process, skipping tryptophan-to-tyrosine radical migration link. This

hypothesis was to be tested on CW, CGW, and CGGW model peptides. Unfortunately,

we were not able to form radical cations of these peptides via Siu's method. Moreover,

use of copper leads to S-S bond formation between oxidized cysteine residues. The same

study has been carried out to investigate potential radical migration of the N-indolyl

tryptophan radical and a cysteine side chain. The lack of reactivity during IMR with

dimethyl disulfide (selective to sulfur-based radicals) at reaction times up to 2000 ms led

to two observations. First, the initial location of the radical was on tryptophan, and

second, there was no radical migration observed between tryptophan and cysteine side

chains at thermal conditions. We expected to initiate radical migration by applying

external collision energy to the system. An evidence of the radical transfer had to be

signature thiol radical fragments (-H2S, -CH2S, -CO2,-HS•). The lack of those fragments

in the CID spectrum, along with the presence of exclusively tryptophan radical fragments

suggested the absence of tryptophan-to-cysteine radical migration in the case of

138

N-indolyl tryptophan radical within a short peptide, although migration between π-radical

cation and cysteine side chain remains to be an open question.

Oxidation of tyrosine to tyrosyl radical is one of the biological functions of

N-indolyl tryptophan radical. We have tested this process on the model tripeptides GYW,

YGW, WGY. The study was based on the tyrosyl radical's ability to lose its side chain

(-106 Da). If the radical is selectively formed on the tryptophan (tyrosine cannot be

nitrosylated using our procedure) the observed neutral loss of 106 Da will be strong

evidence of the radical migration from tryptophan to tyrosine with a further formation of

the glycine-based radical via the tyrosine side chain cleavage. The obtained radicals did

not demonstrate any reactivity towards NO• gas, even at longer reaction times during

IMR experiments, which confirmed the initial radical location on tryptophan (TyrO• does

react with NO•) as well as proved the lack of HAT under thermal conditions. CID

experiments were performed in to supply isolated radicals with additional energy and

initiate radical migration. The signature tyrosine side chain loss was observed in GYW

and YGW fragmentation spectra, and was not present in GWY fragmentation, suggesting

a possibility of the radical migration if the direction of this process is towards the

N-terminus of the peptide. Based on the relatively low peak abundance (of [M-106]+ ion)

and presence of the tryptophan-based radical peaks (such as m/z 117, m/z 143, m/z 187,

m/z 204, m/z 264), we can postulate a partial radical migration between tryptophan and

tyrosine side-chains. As a control experiment, the peptide radicals produced via ternary

copper complex dissociation were examined as well. Since it was previously shown that

139

the radical migration process between tryptophan and tyrosine side chains involve HAT

to the indole nitrogen, which in the π-radical is protonated, we did not expect to see any

migration to occur. Another factor in the experiment is that both tryptophan and tyrosine

can be oxidized by the ternary metal complex, so the initial location of the radical is not

certain, although we can assume that during the tryptophan oxidation the radical will stay

intact, while tyrosine oxidation will lead to the observation of the loss of -106 Da during

CID. The main fragmentation pattern looks similar to that of N-radicals, regarding the

presence of fragments common for the radical site being located on the tryptophan

residue, although the loss of 106 Da was present in the YGW spectrum. This can be

explained by the partial presence of a stable tyrosine-based radical (which does not

spontaneously lose the side chain) along with majority of π-radical. In this case, the loss

of the tyrosine side chain originates from the initially formed tyrosine-based radical.

Alternatively, the radical transfer occurs not only from the N-indolyl radical, but from the

π-radical cation as well. Both scenarios are equally plausible, so additional experiments

need to take place in order to define the correct process.

6.1 Future Work

The results and conclusions from the current work open new opportunities for

future research directions and improvements. Two types of tryptophan-based radicals

were compared by their reactivity at the same reaction conditions. Further studies can

include kinetics experiments with benzeneselenol and di-tert-butyl nitroxide to estimate

140

rates of the proton-coupled electron transfer and electron transfer, respectively, which can

potentially explain kinetics of the radical - antioxidant system interactions.

IRMPD spectroscopy can be potentially very useful in the IR range which shows

hydrogen bonds and N-H bond stretching (3300-3600 cm-1

). Since the N-indolyl radical

in tryptophan-containing peptides is protonated at the N-terminus, a strong hydrogen

bond between the N-terminal hydrogen and the carbonyl oxygen will be most likely

formed. Also, there will be no N-H stretch in indole. As opposed to the N-indolyl radical,

π-radical cation IR spectrum will have N-H bond features of the indole moiety. These

two features can be used to confirm and contrast the position of the radical within

tryptophan and tryptophan-containing small peptides.

Tryptophan-containing peptide radical dissociation pathways need to be estimated

using DFT-calculations. The structure and formation mechanism of the m/z 204 fragment

of the nAW•+

ion remains to be unknown, although preliminary data suggest this ion is a

Cα tryptophan radical. The location of the radical must be confirmed for the future

comparison of its properties with the π-radical cation and N-indolyl radical. As a part of

the future work on tryptophan-containing peptides, acidic amino acids could be

incorporated into the sequence in order to create a hydrogen bond with indole nitrogen

which could potentially change the mechanism of N-indole radical formation and

dissociation.

141

There is still a lot of potential in tryptophan-to-cysteine radical transfer study.

Ternary metal complexes during CID experiments can dissociate by different

mechanisms, formation of the charged radical during dissociation is only one of the

possible outcomes. The radical migration between tryptophan π-radical cation and

cysteine can be further investigated with a condition that an appropriate metal-ligand

combination is found. However, the process of the radical formation on tryptophan might

need to be coupled with a technique to prevent simultaneous oxidation of cysteine and

tryptophan or selective reduction of the S-S bond if one will be formed.

Tryptophan-to-tyrosine radical migration will benefit from the study of the effect

of adjacent amino acid residues on the radical migration kinetics. Hence, varying the

distance between side chains by incorporation of the glycine spacers (or more rigid

prolines) can give some interesting data to consider. The effect of the D-isomers

vs. L-isomers in the solvent-free surround can be studied as well.

The gas-phase ion-molecule reactions study proved to be a robust and reliable

technique with a relatively inexpensive setup. The downside of IMR is its limit to

monitor reactions on a millisecond time scale. Because radical-driven processes are often

faster than that, future studies, aimed to understand the radical migration pathways may

require a gas-phase laser spectroscopy to analyze processes on a nanosecond time scale.

REFERENCES

1. Gerschman, R., Gilbert, D.L., Nye, S.W., Dwyer, P., Fenn, W.O.: Oxygen

poisoning and x-irradiation: a mechanism in common. Sci Aging Knowledge

Environ 2005(17), cp1 (2005)

2. McCord, J.M., Fridovich, I.: The Utility of Superoxide Dismutase in Studying

Free Radical Reactions: I. RADICALS GENERATED BY THE INTERACTION

OF SULFITE, DIMETHYL SULFOXIDE, AND OXYGEN. J. Biol. Chem.

244(22), 6056-6063 (1969)

3. Gutteridge, J.M.C., Halliwell, B.: Free Radicals and Antioxidants in the Year

2000: A Historical Look to the Future. Ann. N. Y. Acad. Sci. 899(1), 136-147

(2000)

4. Davies, M.J., Dean, R.T., Oxford University Press, New York (1997)

5. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.D., Mazur, M., Telser, J.: Free

radicals and antioxidants in normal physiological functions and human disease.

Int. J. Biochem. Cell Biol. 39(1), 44-84 (2007)

6. Dröge, W.: Free radicals in the physiological control of cell function. Physiol.

Rev. 82(1), 47-95 (2002)

7. Knight, J.A.: Review: Free radicals, antioxidants, and the immune system. Ann.

Clin. Lab. Sci. 30(2), 145-158 (2000)

8. Osburn, S., O'Hair, R.A.J., Ryzhov, V.: Gas-Phase Reactivity of Sulfur-Based

Radical Ions of Cysteine Derivatives and Small Peptides. Int. J. Mass spectrom.

316-318,, 133-139 (2012)

143

9. Machlin, L.J., Bendich, A.: Free radical tissue damage: protective role of

antioxidant nutrients. FASEB J. 1(6), 441-445 (1987)

10. Hawkins, C.L., Davies, M.J.: Generation and propagation of radical reactions on

proteins. Biochim. Biophys. Acta 1504(2–3), 196-219 (2001)

11. Davies, M.J.: Singlet oxygen-mediated damage to proteins and its consequences.

Biochem. Biophys. Res. Commun. 305(3), 761-770 (2003)

12. Wagner, B.A., Buettner, G.R., Burns, C.P.: Free Radical-Mediated Lipid

Peroxidation in Cells: Oxidizability Is a Function of Cell Lipid bis-Allylic

Hydrogen Content. Biochemistry 33(15), 4449-4453 (1994)

13. Mylonas, C., Kouretas, D.: Lipid peroxidation and tissue damage. In Vivo 13(3),

295-309 (1998)

14. Cadenas, E., Davies, K.J.A.: Mitochondrial free radical generation, oxidative

stress, and aging. Free Radical Biol. Med. 29(3–4), 222-230 (2000)

15. von Sonntag, C.: Free-radical-induced DNA damage and its repair, Springer,

(2006).

16. Dreher, D., Junod, A.: Role of oxygen free radicals in cancer development. Eur. J.

Can. 32(1), 30-38 (1996)

17. Stocker, R., Keaney, J.F.: Role of oxidative modifications in atherosclerosis.

Physiol. Rev. 84(4), 1381-1478 (2004)

18. Kono, H., Rusyn, I., Yin, M., Gäbele, E., Yamashina, S., Dikalova, A., Kadiiska,

M.B., Connor, H.D., Mason, R.P., Segal, B.H.: NADPH oxidase–derived free

radicals are key oxidants in alcohol-induced liver disease. J. Clin. Invest. 106(7),

867-872 (2000)

144

19. Jenner, P., Dexter, D., Sian, J., Schapira, A., Marsden, C.: Oxidative stress as a

cause of nigral cell death in Parkinson's disease and incidental Lewy body

disease. Ann. Neurol. 32(S1), S82-S87 (1992)

20. Markesbery, W.R.: Oxidative stress hypothesis in Alzheimer's disease. Free

Radical Biol. Med. 23(1), 134-147 (1997)

21. Mahadik, S.P., Mukherjee, S.: Free radical pathology and antioxidant defense in

schizophrenia: a review. Schizophr. Res. 19(1), 1-17 (1996)

22. Logan, D.T., Andersson, J., Sjoberg, B.-M., Nordland, P.: A Glycyl Radical Site

in the Crystal Structure of a Class III Ribonucleotide Reductase. Science

283(5407), 1499-1504 (1999)

23. Becker, A., Kabsch, W.: X-Ray Structure of Pyruvate Formate-Lyase in Complex

with Pyruvate and CoA: How the Enzyme Uses the Cys-418 Thiyl Radical for

Pyruvate Cleavage. J. Biol. Chem. 277(42), 40036-40042 (2002)

24. Ehrenberg, A., Reichard, P.: Electron Spin Resonance of the Iron-Containing

Protein B2 from Ribonucleotide Reductase. J. Biol. Chem. 247(11), 3485-3488

(1972)

25. Sivaraja, M., Goodin, D.B., Smith, M., Hoffman, B.M.: Identification by ENDOR

of Trp191 as the Free-Radical Site in Cytochrome C Peroxidase Compound ES.

Science 245(4919), 738-740 (1989)

26. Easton, C.J.: Free-radical reactions in the synthesis of α-amino acids and

derivatives. Chem. Rev. 97(1), 53-82 (1997)

27. Viehe, H.G., Janousek, Z., Merenyi, R., Stella, L.: The captodative effect. Acc.

Chem. Res. 18(5), 148-154 (1985)

145

28. Hioe, J., Savasci, G., Brand, H., Zipse, H.: The stability of Cα peptide radicals:

Why glycyl radical enzymes? Chem.Eur. J. 17(13), 3781-3789 (2011)

29. Eklund, H., Fontecave, M.: Glycyl radical enzymes: a conservative structural

basis for radicals. Structure 7(11), R257-R262 (1999)

30. Selmer, T., Pierik, A.J., Heider, J.: New glycyl radical enzymes catalysing key

metabolic steps in anaerobic bacteria. Biol. Chem. 386(10), 981-988 (2005)

31. Becker, A., Fritz-Wolf, K., Kabsch, W., Knappe, J., Schultz, S., Wagner, A.V.:

Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase.

Nat. Struct. Mol. Biol. 6(10), 969-975 (1999)

32. O'Brien, J.R., Raynaud, C., Croux, C., Girbal, L., Soucaille, P., Lanzilotta, W.N.:

Insight into the mechanism of the B12-independent glycerol dehydratase from

Clostridium butyricum: preliminary biochemical and structural characterization.

Biochemistry 43(16), 4635-4645 (2004)

33. Vey, J.L., Yang, J., Li, M., Broderick, W.E., Broderick, J.B., Drennan, C.L.:

Structural basis for glycyl radical formation by pyruvate formate-lyase activating

enzyme. Proc. Natl. Acad. Sci. 105(42), 16137-16141 (2008)

34. Frey, M., Rothe, M., Wagner, A., Knappe, J.: Adenosylmethionine-dependent

synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the

glycine C-2 pro-S hydrogen atom. Studies of [2H] glycine-substituted enzyme

and peptides homologous to the glycine 734 site. J. Biol. Chem. 269(17), 12432-

12437 (1994)

35. Lehtio, L., Leppanen, V.-M., Kozarich, J.W., Goldman, A.: Structure of

Escherichia coli pyruvate formate-lyase with pyruvate. Acta Crystallogr. Sect. D

58(12), 2209-2212 (2002)

146

36. Logan, D.T., Andersson, J., Sjöberg, B.-M., Nordlund, P.: A glycyl radical site in

the crystal structure of a class III ribonucleotide reductase. Science 283(5407),

1499-1504 (1999)

37. Leuthner, B., Leutwein, C., Schulz, H., Hörth, P., Haehnel, W., Schiltz, E.,

Schägger, H., Heider, J.: Biochemical and genetic characterization of

benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme

catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28(3),

615-628 (1998)

38. Himo, F.: C–C bond formation and cleavage in radical enzymes, a theoretical

perspective. Biochim. Biophys. Acta 1707(1), 24-33 (2005)

39. Qiao, C., Marsh, E.N.G.: Mechanism of Benzylsuccinate Synthase:

Stereochemistry of Toluene Addition to Fumarate and Maleate. J. Am. Chem. Soc.

127(24), 8608-8609 (2005)

40. Selmer, T., Andrei, P.I.: p-Hydroxyphenylacetate decarboxylase from Clostridium

difficile. Eur. J. Biochem. 268(5), 1363-1372 (2001)

41. Yu, L., Blaser, M., Andrei, P.I., Pierik, A.J., Selmer, T.: 4-Hydroxyphenylacetate

Decarboxylases: Properties of a Novel Subclass of Glycyl Radical Enzyme

Systems†. Biochemistry 45(31), 9584-9592 (2006)

42. Elsden, S., Hilton, M., Waller, J.: The end products of the metabolism of aromatic

amino acids by clostridia. Arch. Microbiol. 107(3), 283-288 (1976)

43. Raynaud, C., Sarçabal, P., Meynial-Salles, I., Croux, C., Soucaille, P.: Molecular

characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium

butyricum. Proc. Natl. Acad. Sci. 100(9), 5010-5015 (2003)

147

44. Reddy, S.G., Wong, K.K., Parast, C.V., Peisach, J., Magliozzo, R.S., Kozarich,

J.W.: Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the

formation of protein-based sulfinyl and peroxyl radicals. Biochemistry 37(2), 558-

563 (1998)

45. Demick, J.M., Lanzilotta, W.N.: Radical SAM activation of the B12-independent

glycerol dehydratase results in formation of 5′-deoxy-5′-(methylthio) adenosine

and not 5′-deoxyadenosine. Biochemistry 50(4), 440-442 (2011)

46. Buckel, W., Golding, B.T.: Radical Enzymes in Anaerobes*. Annu. Rev.

Microbiol. 60, 27-49 (2006)

47. Mulliez, E., Padovani, D., Atta, M., Alcouffe, C., Fontecave, M.: Activation of

class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron

transfer to the iron-sulfur center. Biochemistry 40(12), 3730-3736 (2001)

48. Mulliez, E., Fontecave, M., Gaillard, J., Reichard, P.: An iron-sulfur center and a

free radical in the active anaerobic ribonucleotide reductase of Escherichia coli. J.

Biol. Chem. 268(4), 2296-2299 (1993)

49. Sun, X., Ollagnier, S., Schmidt, P.P., Atta, M., Mulliez, E., Lepape, L., Eliasson,

R., Gräslund, A., Fontecave, M., Reichard, P.: The free radical of the anaerobic

ribonucleotide reductase from Escherichia coli is at glycine 681. J. Biol. Chem.

271(12), 6827-6831 (1996)

50. Stubbe, J., van der Donk, W.A.: Protein radicals in enzyme catalysis. Chem. Rev.

98(2), 705-762 (1998)

51. Tamarit, J., Mulliez, E., Meier, C., Trautwein, A., Fontecave, M.: The Anaerobic

Ribonucleotide Reductase from Escherichia coli : THE SMALL PROTEIN IS AN

ACTIVATING ENZYME CONTAINING A [4Fe-4S]2+

CENTER. J. Biol. Chem.

274(44), 31291-31296 (1999)

148

52. Young, P., Andersson, J., Sahlin, M., Sjöberg, B.-M.: Bacteriophage T4 anaerobic

ribonucleotide reductase contains a stable glycyl radical at position 580. J. Biol.

Chem. 271(34), 20770-20775 (1996)

53. Thibodeaux, C.J., van der Donk, W.A.: Converging on a mechanism for choline

degradation. Proc. Natl. Acad. Sci. 109(52), 21184-21185 (2012)

54. Craciun, S., Balskus, E.P.: Microbial conversion of choline to trimethylamine

requires a glycyl radical enzyme. Proc. Natl. Acad. Sci. 109(52), 21307-21312

(2012)

55. Craciun, S., Marks, J.A., Balskus, E.P.: Characterization of Choline

Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzymes. ACS

Chem. Biol. (2014)

56. King, D.S., Reichard, P.: Mass spectrometric determination of the radical scission

site in the anaerobic ribonucleotide reductase of Escherichia coli. Biochem.

Biophys. Res. Commun. 206(2), 731-735 (1995)

57. Prütz, W.: in Sulfur-Centered Reactive Intermediates in Chemistry and Biology,

Vol. 197 (Eds.: C. Chatgilialoglu, K.-D. Asmus), Springer US, (1990), p. 389-399.

58. Westerlund, K., Berry, B.W., Privett, H.K., Tommos, C.: Exploring amino-acid

radical chemistry: protein engineering and de novo design. Biochim. Biophys.

Acta 1707(1), 103-116 (2005)

59. Whittaker, M.M., Whittaker, J.W.: The active site of galactose oxidase. J. Biol.

Chem. 263(13), 6074-6080 (1988)

60. Ito, N., Phillips, S.E., Stevens, C., Ogel, Z.B., McPherson, M.J., Keen, J.N.,

Yadav, K.D., Knowles, P.F.: Novel thioether bond revealed by a 1.7 Å crystal

structure of galactose oxidase. Nature (1991)

149

61. Minasian, S.G., Whittaker, M.M., Whittaker, J.W.: Stereoselective hydrogen

abstraction by galactose oxidase. Biochemistry 43(43), 13683-13693 (2004)

62. Rogers, M.S., Baron, A.J., McPherson, M.J., Knowles, P.F., Dooley, D.M.:

Galactose Oxidase Pro-Sequence Cleavage and Cofactor Assembly Are Self-

Processing Reactions. J. Am. Chem. Soc. 122(5), 990-991 (2000)

63. Whittaker, J.W.: Free radical catalysis by galactose oxidase. Chem. Rev. 103(6),

2347-2364 (2003)

64. Whittaker, M.M., Kersten, P.J., Nakamura, N., Sanders-Loehr, J., Schweizer,

E.S., Whittaker, J.W.: Glyoxal Oxidase from Phanerochaete chrysosporium Is a

New Radical-Copper Oxidase. J. Biol. Chem. 271(2), 681-687 (1996)

65. Kersten, P.J.: Glyoxal oxidase of Phanerochaete chrysosporium: its

characterization and activation by lignin peroxidase. Proc. Natl. Acad. Sci. 87(8),

2936-2940 (1990)

66. Halfen, J.A., Jazdzewski, B.A., Mahapatra, S., Berreau, L.M., Wilkinson, E.C.,

Que, L., Tolman, W.B.: Synthetic Models of the Inactive Copper(II)−Tyrosinate

and Active Copper(II)−Tyrosyl Radical Forms of Galactose and Glyoxal

Oxidases. J. Am. Chem. Soc. 119(35), 8217-8227 (1997)

67. Migliore, A., Polizzi, N.F., Therien, M.J., Beratan, D.N.: Biochemistry and

Theory of Proton-Coupled Electron Transfer. Chem. Rev. 114(7), 3381-3465

(2014)

68. Minnihan, E.C., Nocera, D.G., Stubbe, J.: Reversible, long-range radical transfer

in E. coli class Ia ribonucleotide reductase. Acc. Chem. Res. 46(11), 2524-2535

(2013)

69. Stubbe, J., Nocera, D.G., Yee, C.S., Chang, M.C.Y.: Radical Initiation in the

Class I Ribonucleotide Reductase: Long-Range Proton-Coupled Electron

Transfer? Chem. Rev. 103(6), 2167-2202 (2003)

150

70. Aubert, C., Mathis, P., Eker, A.P., Brettel, K.: Intraprotein electron transfer

between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans.

Proc. Natl. Acad. Sci. 96(10), 5423-5427 (1999)

71. Erman, J.E., Vitello, L.B.: Yeast cytochrome c peroxidase: mechanistic studies

via protein engineering. Biochim. Biophys. Acta 1597(2), 193-220 (2002)

72. DeGray, J., Lassmann, G., Curtis, J.F., Kennedy, T., Marnett, L., Eling, T.E.,

Mason, R.P.: Spectral analysis of the protein-derived tyrosyl radicals from

prostaglandin H synthase. J. Biol. Chem. 267(33), 23583-23588 (1992)

73. Tsai, A.-l., Hsi, L.C., Kulmacz, R.J., Palmer, G., Smith, W.L.: Characterization of

the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement

and site-directed mutagenesis. J. Biol. Chem. 269(7), 5085-5091 (1994)

74. Ivancich, A., Jouve, H.M., Sartor, B., Gaillard, J.: EPR investigation of compound

I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and

tyrosyl radical intermediates. Biochemistry 36(31), 9356-9364 (1997)

75. Su, C., Sahlin, M., Oliw, E.H.: A protein radical and ferryl intermediates are

generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and

hydroperoxide isomerase activities. J. Biol. Chem. 273(33), 20744-20751 (1998)

76. Babcock, G.T., Barry, B., Debus, R., Hoganson, C., Atamian, M., McIntosh, L.,

Sithole, I., Yocum, C.: Water oxidation in photosystem II: from radical chemistry

to multielectron chemistry. Biochemistry 28(25), 9557-9565 (1989)

77. Meyer, T.J., Huynh, M.H.V., Thorp, H.H.: The Possible Role of Proton‐Coupled

Electron Transfer (PCET) in Water Oxidation by Photosystem II. Angew. Chem.

Int. Ed. 46(28), 5284-5304 (2007)

78. Faller, P., Debus, R.J., Brettel, K., Sugiura, M., Rutherford, A.W., Boussac, A.:

Rapid formation of the stable tyrosyl radical in photosystem II. Proc. Natl. Acad.

Sci. 98(25), 14368-14373 (2001)

151

79. Broderick, J.B., Duffus, B.R., Duschene, K.S., Shepard, E.M.: Radical S-

Adenosylmethionine Enzymes. Chem. Rev. 114(8), 4229-4317 (2014)

80. Horikawa, S., Sasuga, J., Shimizu, K., Ozasa, H., Tsukada, K.: Molecular cloning

and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine

synthetase. J. Biol. Chem. 265(23), 13683-13686 (1990)

81. Stubbe, J.: Ribonucleotide reductases in the twenty-first century. Proc. Natl.

Acad. Sci. 95(6), 2723-2724 (1998)

82. McGuirl, M.A., Dooley, D.M.: Copper-containing oxidases. Curr. Opin. Chem.

Biol. 3(2), 138-144 (1999)

83. Yaropolov, A., Kharybin, A., Emneus, J., Marko-Varga, G., Gorton, L.:

Electrochemical properties of some copper-containing oxidases.

Bioelectrochemistry 40(1), 49-57 (1996)

84. Miller, M.A., Vitello, L., Erman, J.E.: Regulation of Interprotein Electron

Transfer By Trp 191 of Cytochrome c Peroxidase. Biochemistry 34(37), 12048-

12058 (1995)

85. Ivancich, A., Dorlet, P., Goodin, D.B., Un, S.: Multifrequency High-Field EPR

Study of the Tryptophanyl and Tyrosyl Radical Intermediates in Wild-Type and

the W191G Mutant of Cytochrome c Peroxidase. J. Am. Chem. Soc. 123(21),

5050-5058 (2001)

86. Himo, F., Eriksson, L.A.: Theoretical Study of Model Tryptophan Radicals and

Radical Cations: Comparison with Experimental Data of DNA Photolyase,

Cytochrome c Peroxidase, and Ribonucleotide Reductase. J. Phys. Chem. B

101(47), 9811-9819 (1997)

87. Liu, Z., Zhang, M., Guo, X., Tan, C., Li, J., Wang, L., Sancar, A., Zhong, D.:

Dynamic determination of the functional state in photolyase and the implication

for cryptochrome. Proc. Natl. Acad. Sci. 110(32), 12972-12977 (2013)

152

88. Chang, C.-W., Guo, L., Kao, Y.-T., Li, J., Tan, C., Li, T., Saxena, C., Liu, Z.,

Wang, L., Sancar, A.: Ultrafast solvation dynamics at binding and active sites of

photolyases. Proc. Natl. Acad. Sci. 107(7), 2914-2919 (2010)

89. Carell, T., Burgdorf, L.T., Kundu, L.M., Cichon, M.: The mechanism of action of

DNA photolyases. Curr. Opin. Chem. Biol. 5(5), 491-498 (2001)

90. Aubert, C., Vos, M.H., Mathis, P., Eker, A.P., Brettel, K.: Intraprotein radical

transfer during photoactivation of DNA photolyase. Nature 405(6786), 586-590

(2000)

91. Klapper, M.H., Faraggi, M.: Applications of pulse radiolysis to protein chemistry.

Q. Rev. Biophys. 12(4), 465-519 (1979)

92. Sonntag, C.: The Chemical Basis of Radiation Biology, Taylor & Francis, (1987)

93. Mishra, A.K., Chandrasekar, R., Faraggi, M., Klapper, M.H.: Long-range electron

transfer in peptides. Tyrosine reduction of the indolyl radical: reaction

mechanism, modulation of reaction rate, and physiological considerations. J. Am.

Chem. Soc. 116(4), 1414-1422 (1994)

94. Chen, X., Zhang, L., Zhang, L., Wang, J., Liu, H., Bu, Y.: Proton-Regulated

Electron Transfers from Tyrosine to Tryptophan in Proteins: Through-Bond

Mechanism versus Long-Range Hopping Mechanism. J. Phys. Chem. B 113(52),

16681-16688 (2009)

95. Han, W.-G., Noodleman, L.: DFT calculations for intermediate and active states

of the diiron center with a tryptophan or tyrosine radical in Escherichia coli

ribonucleotide reductase. Inorg. Chem. 50(6), 2302-2320 (2011)

96. Shih, C., Museth, A.K., Abrahamsson, M., Blanco-Rodriguez, A.M., Di Bilio,

A.J., Sudhamsu, J., Crane, B.R., Ronayne, K.L., Towrie, M., Vlček, A.:

Tryptophan-accelerated electron flow through proteins. Science 320(5884), 1760-

1762 (2008)

153

97. Pogni, R., Baratto, M.C., Teutloff, C., Giansanti, S., Ruiz-Dueñas, F.J.,

Choinowski, T., Piontek, K., Martínez, A.T., Lendzian, F., Basosi, R.: A

Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from

Pleurotus eryngii. J. Biol. Chem. 281(14), 9517-9526 (2006)

98. Goblirsch, B., Streit, B., DuBois, J., Wilmot, C.: Structural features promoting

dioxygen production by Dechloromonas aromatica chlorite dismutase. J. Biol.

Inorg. Chem. 15(6), 879-888 (2010)

99. Veesler, S., Furuta, K., Horiuchi, H., Hiratsuka, H., Ferté, N., Okutsu, T.: Crystals

from Light: Photochemically Induced Nucleation of Hen Egg-White Lysozyme.

Cryst. Growth Des. 6(7), 1631-1635 (2006)

100. Grossweiner, L.I., Kaluskar, A.G., Baugher, J.F.: Flash Photolysis of Enzymes.

Int. J. Radiat. Biol. 29(1), 1-16 (1976)

101. Kumagai, I., Sunada, F., Takeda, S., Miura, K.: Redesign of the substrate-binding

site of hen egg white lysozyme based on the molecular evolution of C-type

lysozymes. J. Biol. Chem. 267(7), 4608-4612 (1992)

102. Flossmann, W., Westhof, E.: Action of ionizing radiation on protein. II. Radical

formation in L-tryptophan-HCl. Radiat. Res. 73(1), 75-85 (1978)

103. DeFelippis, M.R., Murthy, C.P., Klapper, M.H., Broitman, F., Weinraub, D.,

Faraggi, M.: Electrochemical properties of tyrosine phenoxy and tryptophan

indolyl radicals in peptides and amino acid analogues. J. Phys. Chem. 95(8),

3416-3419 (1991)

104. Connor, H.D., Sturgeon, B.E., Mottley, C., Sipe, H.J., Mason, R.P.: l-Tryptophan

Radical Cation Electron Spin Resonance Studies: Connecting Solution-Derived

Hyperfine Coupling Constants with Protein Spectral Interpretations. J. Am. Chem.

Soc. 130(20), 6381-6387 (2008)

154

105. Lendzian, F., Sahlin, M., MacMillan, F., Bittl, R., Fiege, R., Pötsch, S., Sjöberg,

B.-M., Gräslund, A., Lubitz, W., Lassmann, G.: Electronic Structure of Neutral

Tryptophan Radicals in Ribonucleotide Reductase Studied by EPR and ENDOR

Spectroscopy. J. Am. Chem. Soc. 118(34), 8111-8120 (1996)

106. Walden, S.E., Wheeler, R.A.: Distinguishing Features of Indolyl Radical and

Radical Cation: Implications for Tryptophan Radical Studies. J. Phys. Chem.

100(5), 1530-1535 (1996)

107. Jovanovic, S.V., Simic, M.G.: Repair of tryptophan radicals by antioxidants. Free

Radic. Biol. Med. 1(2), 125-129 (1985)

108. Carugo, O., Carugo, K.D.: When X-rays modify the protein structure: radiation

damage at work. Trends Biochem. Sci. 30(4), 213-219 (2005)

109. Li, W., Zheng, R., Jia, Z., Zou, Z., Lin, N.: Repair effect of phenylpropanoid

glycosides on thymine radical anion induced by pulse radiolysis. Biophys. Chem.

67(1–3), 281-286 (1997)

110. Davies, M.J.: The oxidative environment and protein damage. Biochim. Biophys.

Acta 1703(2), 93-109 (2005)

111. Vásquez-Vivar, J., Whitsett, J., Martásek, P., Hogg, N., Kalyanaraman, B.:

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and

characterization of the pteridine radical. Free Radical Biol. Med. 31(8), 975-985

(2001)

112. Hawkins, C., Davies, M.: EPR studies on the selectivity of hydroxyl radical attack

on amino acids and peptides. J. Chem. Soc., Perkin Trans. 2 (12), 2617-2622

(1998)

113. Prütz, W.A., Butler, J., Land, E.J., Swallow, A.J.: The role of sulphur peptide

functions in free radical transfer: a pulse radiolysis study. Int. J. Radiat. Biol.

55(4), 539-556 (1989)

155

114. Page, C.C., Moser, C.C., Chen, X., Dutton, P.L.: Natural engineering principles of

electron tunnelling in biological oxidation-reduction. Nature 402(6757), 47-52

(1999)

115. Yang, L., Nelson, R.S., Benjdia, A., Lin, G., Telser, J., Stoll, S., Schlichting, I.,

Li, L.: A radical transfer pathway in spore photoproduct lyase. Biochemistry

52(18), 3041-3050 (2013)

116. Prütz, W.A., Siebert, F., Butler, J., Land, E.J., Menez, A., Montenay-Garestier, T.:

Charge transfer in peptides: Intramolecular radical transformations involving

methionine, tryptophan and tyrosine. Biochim. Biophys. Acta 705(2), 139-149

(1982)

117. Papayannopoulos, I.A.: The interpretation of collision-induced dissociation

tandem mass spectra of peptides. Mass Spectrom. Rev. 14(1), 49-73 (1995)

118. Zubarev, R.A., Kelleher, N.L., McLafferty, F.W.: Electron Capture Dissociation

of Multiply Charged Protein Cations. A Nonergodic Process. J. Am. Chem. Soc.

120(13), 3265-3266 (1998)

119. Kelleher, N.L.: Peer reviewed: Top-down proteomics. Anal. Chem. 76(11), 196

A-203 A (2004)

120. Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., Whitehouse, C.M.: Electrospray

ionization for mass spectrometry of large biomolecules. Science 246(4926), 64-71

(1989)

121. Budnik, B.A., Haselmann, K.F., Zubarev, R.A.: Electron detachment dissociation

of peptide di-anions: an electron–hole recombination phenomenon. Chem. Phys.

Lett. 342(3–4), 299-302 (2001)

122. Syka, J.E.P., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and

protein sequence analysis by electron transfer dissociation mass spectrometry.

Proc. Natl. Acad. Sci. U. S. A. 101(26), 9528-9533 (2004)

156

123. Tureček, F., Julian, R.R.: Peptide Radicals and Cation Radicals in the Gas Phase.

Chem. Rev. 113(8), 6691-6733 (2013)

124. Siu, C.-K., Ke, Y., Orlova, G., Hopkinson, A., Siu, K.W.M.: Dissociation of the

N-Cα bond and competitive formation of the [zn−H]•+

and [cn+2H]+ product ions

in radical peptide ions containing tyrosine and tryptophan: The influence of

proton affinities on product formation. J. Am. Soc. Mass Spectrom. 19(12), 1799-

1807 (2008)

125. Sun, Q., Nelson, H., Ly, T., Stoltz, B.M., Julian, R.R.: Side Chain Chemistry

Mediates Backbone Fragmentation in Hydrogen Deficient Peptide Radicals. J.

Proteome Res. 8(2), 958-966 (2008)

126. Ryzhov, V., Lam, A.K.Y., O'Hair, R.A.J.: Gas-Phase Fragmentation of Long-

Lived Cysteine Radical Cations Formed via NO Loss from Protonated S-

Nitrosocysteine. J. Am. Soc. Mass Spectrom. 20(6), 985-995 (2009)

127. Song, T., Hao, Q., Law, C.-H., Siu, C.-K., Chu, I.: Novel Cβ–Cγ Bond Cleavages

of Tryptophan-Containing Peptide Radical Cations. J. Am. Soc. Mass Spectrom.

23(2), 264-273 (2012)

128. Song, T., Ma, C.-Y., Chu, I.K., Siu, C.-K., Laskin, J.: Mechanistic Examination of

Cβ–Cγ Bond Cleavages of Tryptophan Residues during Dissociations of

Molecular Peptide Radical Cations. J. Phys. Chem. A (2012)

129. Zhang, X., Julian, R.: Radical Additions to Aromatic Residues in Peptides

Facilitate Unexpected Side Chain and Backbone Losses. J. Am. Soc. Mass

Spectrom. 25(4), 626-635 (2014)

130. Mädler, S., Kai-Chi Lau, J., Williams, D., Wang, Y., Saminathan, I.S., Zhao, J.,

Siu, K.W.M., Hopkinson, A.C.: Fragmentation of Peptide Radical Cations

Containing a Tyrosine or Tryptophan Residue: Structural Features That Favor

Formation of [x(n–1) H]•+

and [z(n–1)

+H]•+

Ions. J. Phys. Chem. B 118(23), 6123-

6133 (2014)

157

131. Bagheri-Majdi, E., Ke, Y., Orlova, G., Chu, I.K., Hopkinson, A.C., Siu, K.W.M.:

Copper-Mediated Peptide Radical Ions in the Gas Phase. J. Phys. Chem. B

108(30), 11170–11181 (2004)

132. Barlow, C.K., McFadyen, W.D., O'Hair, R.A.J.: Formation of Cationic Peptide

Radicals by Gas-Phase Redox Reactions with Trivalent Chromium, Manganese,

Iron, and Coblat complexes. J. Am. Chem. Soc. 127(16), 6109-6115 (2005)

133. Barlow, C.K., Moran, D., Radom, L., McFadyen, W.D., O'Hair, R.A.J.: Metal-

Mediated Formation of Gas-Phase Amino Acid Radical Cations. J. Phys. Chem. A

110(27), 8304-8315 (2006)

134. Lias, S.G.: Gas-phase ion and neutral thermochemistry. J. Phys. Chem. Ref Data

17(1)(1988)

135. Hao, G., Gross, S.S.: Electrospray Tandem Mass Spectrometry Analysis of S- and

N-Nitrosopetdies: Facile Loss of NO and Radical-Induced Fragmentation. J. Am.

Soc. Mass Spectrom. 17(12), 1725-1730 (2006)

136. Knudsen, E.R., Julian, R.R.: Fragmentation Chemistry Observed in Hydrogen

Deficient Radical Peptides Generated from N-Nitrosotryptophan Residues. Int. J.

Mass spectrom. 294(2-3), 83-87 (2010)

137. Ly, T., Julian, R.R.: Residue-Specific Radical-Directed Dissociation of Whole

Proteins in the Gas Phase. J. Am. Chem. Soc. 130(1), 351-358 (2007)

138. Moore, B.N., Blanksby, S.J., Julian, R.R.: Ion-molecule reactions reveal facile

radical migration in peptides. Chem. Commun. (33), 5015-5017 (2009)

139. Laskin, J., Yang, Z., Ng, C., Chu, I.K.: Fragmentation of α-radical cations of

arginine-containing peptides. J. Am. Soc. Mass Spectrom. 21(4), 511-521 (2010)

158

140. Chu, I.K., Zhao, J., Xu, M., Siu, S.O., Hopkinson, A.C., Siu, K.W.M.: Are the

Radical Centers in Peptide Radical Cations Mobile? The Generation,

Tautomerism, and Dissociation of Isomeric α-Carbon-Centered Triglycine

Radical Cations in the Gas Phase. J. Am. Chem. Soc. 130(25), 7862-7872 (2008)

141. Simon, S., Sodupe, M., Bertran, J.: Isomerization versus Fragmentation of

Glycine Radical Cation in Gas Phase. J. Phys. Chem. A 106(23), 5697-5702

(2002)

142. ek, F., Carpenter, F.H., Polce, M.J., Wesdemiotis, C.: Glycyl Radical Is a

Stable Species in the Gas Phase. J. Am. Chem. Soc. 121(34), 7955-7956 (1999)

143. Osburn, S., Berden, G., Oomens, J., O‟Hair, R.J., Ryzhov, V.: S-to-αC Radical

Migration in the Radical Cations of Gly-Cys and Cys-Gly. J. Am. Soc. Mass

Spectrom. 23(6), 1019-1023 (2012)

144. Chu, I.K., Rodriquez, C.F., Lau, T.-C., Hopkinson, A.C., Siu, K.W.M.: Molecular

Radical Cations of Oligopeptides. J. Phys. Chem. B 104(15), 3393-3397 (2000)

145. Hopkinson, A.C., Siu, K.W.M.: in Principles of Mass Spectrometry Applied to

Biomolecules (Eds.: J. Laskin, C. Lifshitz), John Wiley & Sons, inc., Hoboken, p.

301-335 (2006)

146. Laskin, J., Yang, Z., Chu, I.K.: Energetics and Dynamics of Electron Transfer and

Proton Transfer in Dissociation of MetalIII

(salen)−Peptide Complexes in the Gas

Phase. J. Am. Chem. Soc. 130(10), 3218-3230 (2008)

147. Pyatkivskyy, Y., Ryzhov, V.: Coupling of ion-molecule reactions with liquid

chromatography on a quadrupole ion trap mass spectrometer. Rapid Commun.

Mass Spectrom. 22(8), 1288-1294 (2008)

148. Masterson, D.S., Yin, H., Chacon, A., Hachey, D.L., Norris, J.L., Porter, N.A.:

Lysine Peroxycarbamates: Free Radical-Promoted Peptide Cleavage. J. Am.

Chem. Soc. 126(3), 720-721 (2004)

159

149. Wee, S., Mortimer, A., Moran, D., Wright, A., Barlow, C.K., O'Hair, R.A.J.,

Radom, L., Easton, C.J.: Gas-Phase Regiocontrolled Generation of Charged

Amino Acid and Peptide Radicals. Chem. Commun., 4233-4235 (2006)

150. Liu, Z., Julian, R.R.: Deciphering the Peptide Iodination Code: Influence on

Subsequent Gas-Phase Radical Generation with Photodissociation ESI-MS. J.

Am. Soc. Mass Spectrom. 20(6), 965-971 (2009)

151. Laskin, J., Yang, Z., Lam, C., Chu, I.K.: Charge-Remote Fragmentation of Odd-

Electron Peptdie Ions. Anal. Chem. 79(17), 6607-6614 (2007)

152. Hopkinson, A.C.: Radical Cations of Amino Acids and Peptides: Structures and

Stabilities. Mass Spectrom. Rev. 28(4), 655-671 (2009)

153. Siu, C.-K., Ke, Y., Guo, Y., Hopkinson, A.C., Siu, K.W.M.: Dissociations of

copper(ii)-containing complexes of aromatic amino acids: radical cations of

tryptophan, tyrosine, and phenylalanine. Phys. Chem. Chem. Phys. 10(38), 5908-

5918 (2008)

154. Ke, Y., Aribi, H.E., Siu, C.-K., Siu, K.W.M., Hopkinson, A.C.: A comparison of

the fragmentation pathways of [CuII(Ma)(Mb)]

•2+ complexes where Ma and Mb are

peptides containing either a tryptophan or a tyrosine residue. Rapid Commun.

Mass Spectrom. 24(23), 3485-3492 (2010)

155. Song, T., Ng, D.C.M., Quan, Q., Siu, C.-K., Chu, I.K.: Arginine-Facilitated α-

and π-Radical Migrations in Glycylarginyltryptophan Radical Cations. Chem.

Asian. J. 6(3), 888-898 (2011)

156. Xu, M., Song, T., Quan, Q., Hao, Q., Fang, D.-C., Siu, C.-K., Chu, I.K.: Effect of

the N-terminal basic residue on facile Cα-C bond cleavages of aromatic-

containing peptide radical cations. Phys. Chem. Chem. Phys. 13(13), 5888-5896

(2011)

160

157. Ng, D.C.M., Song, T., Siu, S.O., Siu, C.K., Laskin, J., Chu, I.K.: Formation,

Isomerization, and Dissociation of α-Carbon-Centered and π-Centered

Glycylglycyltryptophan Radical Cations. J. Phys. Chem. B 114(6), 2270-2280

(2010)

158. Joly, L., Antoine, R., Allouche, A.-R., Dugourd, P.: Formation and Spectroscopy

of a Tryptophan Radical Containing Peptide in the Gas Phase. J. Am. Chem. Soc.

130(42), 13832-13833 (2008)

159. Steill, J.D., Zhao, J., Siu, C.-K., Ke, Y., Verkerk, U., Oomens, J., Dunbar, R.,

Hopkinson, A.C., Siu, K.W.M.: Structure of the Observable Histidine Radical

Cation in the Gas-Phase: A Captodative α-radical Ion. Angew. Chem. Int. Ed.

47(50), 9666-9668 (2008)

160. Sinha, R.K., Maitre, P., Piccirillo, S., Chiavarino, B., Crestoni, M.E., Fornarini,

S.: Cysteine Radical Cation: A Distonic Structure Probed by Gas Phase IR

Spectroscopy. Phys. Chem. Chem. Phys. 12(33), 9794-9800 (2010)

161. Osburn, S., Berden, G., Oomens, J., O'Hair, R.A.J., Ryzhov, V.: Structure and

Reactivity of the N-AcetylCysteine Radical Cation and Anion: Does Radical

Migration occur? J. Am. Soc. Mass Spectrom. 22(10), 1794-1803 (2011)

162. Osburn, S., Steill, J.D., Oomens, J., O'Hair, R.A.J., Van Stipdonk, M., Ryzhov,

V.: Structure and Reactivity of the Cysteine Methyl Ester Radical Cation. Chem.

Eur. J. 17(3), 873-879 (2011)

163. Bellina, B., Compagnon, I., Houver, S., Maître, P., Allouche, A.R., Antoine, R.,

Dugourd, P.: Spectroscopic Signatures of Peptides Containing Tryptophan

Radical Cations. Angew. Chem. Int. Ed. 50(47), 11430–11432 (2011)

164. Pyatkivskyy, Y., Ryzhov, V.: Coupling of Ion-Molecule Reactions to Liquid

Chromatography on a Quadrupole Ion Trap Mass Spectrometer. Rapid Commun.

Mass Spectrom. 22(8), 1288-1294 (2008)

161

165. Oepts, D., van der Meer, A.F.G., van Amersfoort, P.W.: The Free-Electron-Laser

user facility FELIX. Infrared Phys. Technol. 36(1), 297-308 (1995)

166. Polfer, N.C., Oomens, J.: Reaction products in mass spectrometry elucidated with

infrared spectroscopy. Phys. Chem. Chem. Phys. 9(29), 3804-3817 (2007)

167. M. J. Frisch, G.W.T., H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.

Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji,

M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L.

Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,

T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J.

E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N.

Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C.

Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E.

Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E.

Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L.

Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.

Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J.

Cioslowski, and D. J. Fox. Gaussian, Inc., Wallingford CT (2009)

168. Polfer , N.C., Oomens, J., Dunbar, R.C.: Alkali Metal Complexes of the

Dipeptides PheAla and AlaPhe: IRMPD Spectroscopy. Phys. Chem. Chem. Phys.

9(4), 579-589 (2008)

169. Oomens, J., Tielens, A.G.G.M., Sartakov, B.G., Helden, G.v., Meijer, G.:

Laboratory Infrared Spectroscopy of Cationic Polycyclic Aromatic Hydrocarbon

Molecules. Astrophys. J. 591(2), 968-985 (2003)

170. Polfer, N.C., Oomens, J., Dunbar, R.C.: IRMPD spectroscopy of metal-

ion/tryptophan complexes. Phys. Chem. Chem. Phys. 8(23), 2744-2751 (2006)

171. Sinclair, W.E., Pratt, D.W.: Structure and vibrational dynamics of aniline and

aniline--Ar from high resolution electronic spectroscopy in the gas phase. J.

Chem. Phys. 105(18), 7942-7956 (1996)

162

172. Piest, H., Helden, G.v., Meijer, G.: Infrared spectroscopy of jet-cooled neutral and

ionized aniline--Ar. J. Chem. Phys. 110(4), 2010-2015 (1999)

173. Bakker, J.M., Aleese, L.M., Meijer, G., von Helden, G.: Fingerprint IR

Spectroscopy to Probe Amino Acid Conformations in the Gas Phase. Phys. Rev.

Lett. 91(20), 203003 (2003)

174. Mino, W.K., Gulyuz, K., Wang, D., Stedwell, C.N., Polfer, N.C.: Gas-Phase

Structure and Dissociation Chemistry of Protonated Tryptophan Elucidated by

Infrared Multiple-Photon Dissociation Spectroscopy. J. Phys. Chem. Lett 2(4),

299-304 (2011)

175. Walden, S.E., Wheeler, R.A.: Structural and vibrational analysis of indolyl radical

and indolyl radical cation from density functional methods. J. Chem. Soc. Perkin

Trans. II (12), 2663-2672 (1996)

176. Crespo, A., Turjanski, A.G., Estrin, D.o.A.: Electronic spectra of indolyl radicals:

a time-dependent DFT study. Chem. Phys. Lett. 365(1–2), 15-21 (2002)

177. Piatkivskyi, A., Osburn, S., Jaderberg, K., Grzetic, J., Steill, J., Oomens, J., Zhao,

J., Lau, J.-C., Verkerk, U., Hopkinson, A., Siu, K.W.M., Ryzhov, V.: Structure

and Reactivity of the Distonic and Aromatic Radical Cations of Tryptophan. J.

Am. Soc. Mass Spectrom. 24(4), 513-523 (2013)

178. Bagheri-Majdi, E., Ke, Y., Orlova, G., Chu, I.K., Hopkinson, A.C., Siu, K.W.M.:

Copper-Mediated Peptide Radical Ions in the Gas Phase. The Journal of Physical

Chemistry B 108(30), 11170-11181 (2004)

179. Kang, H., Dedonder-Lardeux, C., Jouvet, C., Martrenchard, S., Gregoire, G.,

Desfrancois, C., Schermann, J.P., Barat, M., Fayeton, J.A.: Photo-induced

dissociation of protonated tryptophan TrpH+: A direct dissociation channel in the

excited states controls the hydrogen atom loss. Phys. Chem. Chem. Phys. 6(10),

2628-2632 (2004)

163

180. Knudsen, E.R., Julian, R.R.: Fragmentation chemistry observed in hydrogen

deficient radical peptides generated from N-nitrosotryptophan residues.

International Journal of Mass Spectrometry 294(2–3), 83-87 (2010)

181. Bray, T.M., Kubow, S.: Involvement of free radicals in the mechanism of 3-

methylindole-induced pulmonary toxicity: an example of metabolic activation in

chemically induced lung disease. Environ. Health Perspect. 64, 61 (1985)

182. Bray, T.M., Emmerson, K.S.: Putative mechanisms of toxicity of 3-methylindole:

from free radical to pneumotoxicosis. Annu. Rev. Pharmacol. Toxicol. 34(1), 91-

115 (1994)

183. Gregersen, J.A., Turecek, F.: Mass-spectrometric and computational study of

tryptophan radicals (Trp+H)• produced by collisional electron transfer to

protonated tryptophan in the gas phase. Phys. Chem. Chem. Phys. 12(41), 13434-

13447 (2010)

184. Wee, S., O‟Hair, R.A.J., McFadyen, W.D.: Comparing the gas-phase

fragmentation reactions of protonated and radical cations of the tripeptides GXR.

Int. J. Mass Spectrom. 234(1–3), 101-122 (2004)

185. Chu, I.K., Lam, C.N.W., Siu, S.O.: Facile Generation of Tripeptide Radical

Cations In Vacuo via Intramolecular Electron Transfer in CuII Tripeptide

Complexes Containing Sterically Encumbered Terpyridine Ligands. J. Am. Soc.

Mass Spectrom. 16(5), 763-771 (2005)

186. Antoine, R., Dugourd, P.: Visible and ultraviolet spectroscopy of gas phase

protein ions. Phys. Chem. Chem. Phys. 13(37), 16494-16509 (2011)

187. Jovanovic, S.V., Harriman, A., Simic, M.G.: Electron-transfer reactions of

tryptophan and tyrosine derivatives. J. Phys. Chem. 90(9), 1935-1939 (1986)

188. Zhao, J., Siu, K.W.M., Hopkinson, A.C.: Glutathione radical cation in the gas

phase; generation, structure and fragmentation. Org. Biomol. Chem. 9(21), 7384-

7392 (2011)

APPENDICES

APPENDICES

APPENDIX A

CONTROL MASS SPECTRA FOR THE AW AND WA PEPTIDES

165

CID spectrum of protonated AW peptide.

166

CID spectrum of protonated WA peptide.

APPENDIX B

IRMPD SPECTRA FOR THE nAW AND nWA PEPTIDE RADICALS

168

IRMPD spectra for nWA.

169

IRMPD spectra for nAW.

APPENDIX C

CID MASS SPECTRA OF GW, WG AND GGW RADICAL CATIONS

AND CONTROLS

171

CID fragmentation of a) protonated GW, b) πGW

•+, c) nGW

•+ radicals.

172

CID fragmentation of a) protonated WG, b) nWG

•+ radicals.

173

CID fragmentation of a) protonated GGW, b) πGGW•+

, c) nGGW•+

, d) G•GW

+ radicals.

abstract gas-phase chemistry of tryptophan … · 50 2.8 comparison of experimental irmpd (black...

Documents