ABSTRACT GAS-PHASE CHEMISTRY OF TRYPTOPHAN … · 50 2.8 Comparison of experimental IRMPD (black...
Transcript of 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.
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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 Experimental Section .............................................................................................. 63
3.2.1 Chemicals and Reagents ................................................................................... 63
3.2.2 Mass Spectrometry ........................................................................................... 63
3.3 Results and Discussion ............................................................................................ 64
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 Experimental Section .............................................................................................. 90
4.2.1 Chemicals and Reagents ................................................................................... 90
4.2.2 Mass Spectrometry: CID and Ion-Molecule Rreactions .................................. 90
4.2.3 Infrared Multiple Photon Dissociation Spectroscopy ...................................... 91
4.3 Results and Discussion ............................................................................................ 92
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
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................ 53
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................................................................................... 57
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................................................................................... 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.
3.2 Experimental Section
3.2.1 Chemicals and Reagents
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
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 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
. The nebulizer gas, needle voltage, and
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 Results and Discussion
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
4.2 Experimental Section
4.2.1 Chemicals and Reagents
All chemicals and reagents were used as received without any further purification.
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
. 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)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 Results and Discussion
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
All chemicals and reagents were used as received without any further purification.
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
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 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
. The nebulizer gas,
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
All chemicals and reagents were used as received without any further purification.
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
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 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
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. 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
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.
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.
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