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Interactions of chiral ions and molecules in gasphaseTowards an understanding of chiral recognition mechanismOleksii Rebrov
Academic dissertation for the Degree of Doctor of Philosophy in Physics at StockholmUniversity to be publicly defended on Tuesday 16 October 2018 at 10.00 in sal FB54, AlbaNovauniversitetscentrum, Roslagstullsbacken 21.
AbstractThis thesis comprises the research related to interactions of enantiopure amino acids with chiral and achiral molecules in gasphase. The investigation of the mechanism responsible for chiral discrimination is of the special interest in this work. Anelectrospray ion source platform (Stockholm University), quadrupole time-of-flight mass spectrometer (University of Oslo)and Fourier-transform ion cyclotron resonance mass spectrometer in combination with OPO laser (Centre Laser Infrarouged'Orsay (CLIO), France) have been used in our studies. Results of experiments on collisions of enantiopure amino acids,namely phenylalanine (Phe), tryptophan (Trp), and methionine (Met) with chiral and achiral targets in high and low energyregimes are presented. The fragmentation process is discussed in detail and compared with generally accepted models ofamino acid fragmentation. Formation of proton bound diastereomeric adducts of amino acid and chiral alcohols (2-butanoland 1-phenylethanol) in single collisions is reported. The emphasis was given to reveal stereochemical effects in abovementioned reactions. The structure and vibrational properties of diastereomeric dimers of tryptophan studied using infraredmultiphoton dissociation (IRMPD) spectrometry are presented. Structures and energies of most stable conformers obtainedwith quantum chemical calculations are described and compared to the experimental data. The stereo-dependent featuresare underlined and the chiral discrimination using IRMPD is addressed.
Stockholm 2018http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-159310
ISBN 978-91-7797-354-6ISBN 978-91-7797-355-3
Department of Physics
Stockholm University, 106 91 Stockholm
INTERACTIONS OF CHIRAL IONS AND MOLECULES IN GASPHASE
Oleksii Rebrov
Interactions of chiral ions andmolecules in gas phase
Towards an understanding of chiral recognition mechanism
Oleksii Rebrov
©Oleksii Rebrov, Stockholm University 2018 ISBN print 978-91-7797-354-6ISBN PDF 978-91-7797-355-3 Printed in Sweden by Universitetsservice US-AB, Stockholm 2018
I dedicate this work to myfamily
i
Abstract
This thesis comprises the research related to interactions of enantiopure amino acids with
chiral and achiral molecules in gas phase. The investigation of the mechanism responsible
for chiral discrimination is of the special interest in this work. An electrospray ion source
platform (Stockholm University), quadrupole time-of-flight mass spectrometer (University
of Oslo) and Fourier-transform ion cyclotron resonance mass spectrometer in combination
with OPO laser (Centre Laser Infrarouge d'Orsay (CLIO), France) have been used in our
studies. Results of experiments on collisions of enantiopure amino acids, namely
phenylalanine (Phe), tryptophan (Trp), and methionine (Met) with chiral and achiral
targets in high- and low-energy regimes are presented. The fragmentation process is
discussed in detail and compared with generally accepted models of amino acid
fragmentation. Formation of proton bound diastereomeric adducts of amino acid and chiral
alcohols (2-butanol and 1-phenylethanol) in single collisions is reported. The emphasis was
given to reveal stereochemical effects in above mentioned reactions. The structure and
vibrational properties of diastereomeric dimers of tryptophan studied using infrared
resonant multiple photon dissociation (IRMPD) spectrometry are presented. Structures
and energies of most stable conformers obtained with quantum chemical calculations are
described and compared to the experimental data. The stereo-dependent features are
underlined and the chiral discrimination using IRMPD is addressed.
ii
Sammanfattning
Avhandlingen omfattar forskning relaterad till interaktioner mellan enantiomera, kirala
aminosyror och kirala och akirala molekyler i gasfas. Undersökningen av mekanismen som
är ansvarig för kiral diskriminering är av särskilt intresse för detta arbete. En
elektrosprajjonkälleplattform (Stockholms universitet), löptidsmasspektrometer
(University of Oslo), och en Fourier-transformbaserad
joncyklotronresonansmasspektrometer i kombination med OPO-laser (Center Laser
Infrarouge d'Orsay (CLIO), Frankrike) har använts i våra studier. Resultat av experiment
på hög- och lågenergikollisioner av enantiomera aminosyror, nämligen fenylalanin (Phe),
tryptofan (Trp) och metionin (Met), med kirala och akirala mål presenteras.
Fragmenteringsprocessen diskuteras i detalj och jämförs med allmänt accepterade
modeller av aminosyrafragmentation. Bildning av protonbundna diastereomera addukter
av aminosyra och kirala alkoholer (2-butanol och 1-fenyletanol) vid enskilda kollisioner
rapporteras. Tonvikten är för att avslöja stereokemiska effekter i ovan nämnda reaktioner.
Strukturen och vibrationsegenskaperna hos diastereomera dimerer av tryptofan studerade
med infraröd multifotondissociation-spektrometri presenteras. Strukturer och energier av
de mest stabila konformatorerna som erhållits med kvantkemiska beräkningar beskrivs och
jämförs med experimentella data. De stereo-beroende egenskaperna är poängterade och
den kirala diskrimineringen med användning av IRMPD är adresserad.
iii
List of Papers
THE FOLLOWING PAPERS ARE INCLUDED IN THIS THESIS
PAPER I: High-Energy Collisions of Protonated Enantiopure Amino
Acids With a Chiral Target Gas
Kostiantyn Kulyk, Oleksii Rebrov, Mark H. Stockett, John D. Alexander, Henning
Zettergren, Henning T. Schmidt, Richard D. Thomas, Henrik Cederquist, Mats
Larsson, International Journal of Mass Spectrometry, 388, 2015, 59–64.
DOI:10.1016/j.ijms.2015.08.010
PAPER II: Low-Energy Collisions of Protonated Enantiopure Amino
Acids with Chiral Target Gases
Kostiantyn Kulyk, Oleksii Rebrov, Mauritz Ryding, Richard D. Thomas, Einar
Uggerud, Mats Larsson, Journal of The American Society for Mass Spectrometry,
28, 2017, 2686–2691.
DOI: 10.1007/s13361-017-1796-7
PAPER III: Chirally Sensitive Collision Induced Dissociation of Proton-
Bound Diastereomeric Complexes of Tryptophan and 2-Butanol
Oleksii Rebrov, Kostiantyn Kulyk, Mauritz Ryding, Richard D. Thomas, Einar
Uggerud, Mats Larsson, Chirality, 29, 2017, 115–119.
DOI: 10.1002/chir.22679
PAPER IV: Non-covalently Bonded Diastereomeric Adducts of Amino
Acids and (S)-1-Phenylethanol in Low-energy Dissociative Collisions
Oleksii Rebrov, Mauritz Ryding, Richard D. Thomas, Einar Uggerud, Mats
Larsson. Journal of The American Society for Mass Spectrometry. Submitted.
PAPER V: IRMPD Spectroscopy of Protonated Tryptophan
Diastereomers
Oleksii Rebrov, Mathias Poline, Philippe Maitre, Estelle Loire, Vasyl Yatsyna, Mats
Larsson, Vitali Zhaunerchyk. In manuscript.
iv
PUBLICATIONS NOT INCLUDED IN THIS THESIS
PAPER A: Cooper Minimum Photoelectron Dynamics Beyond the One-
Particle Picture in Chiral Molecules
L. Schio, M. Alagia, D. Toffoli, P. Decleva, O. Rebrov, V. Zhaunerchyk, M. Larsson,
S. Falcinelli, D. Catone, S. Turchini, N. Zema, F. Salvador, P. Bertoch, D. Benedetti,
S. Stranges. In manuscript.
PAPER B: Fragmentation of Positively-Charged Biological Ions
Activated with a Beam of High-Energy Cations
K. Chingin, A. Makarov, E. Denisov, O. Rebrov, R. A. Zubarev, Analytical
Chemistry, 86 (1), 2014, 372–379.
Reprints were made with permission from the publishers.
v
Author’s contribution
PAPER I: Major part of experiments and data processing. Part of the data
analysis. Discussion of the results and manuscript.
PAPER II: Major part of experiments, data processing and data analysis. Part of
writing.
PAPER III: Suggested and performed major part of experiments, data analysis
and major part of the writing.
PAPER IV: Major part of experiments and data processing, data analysis and
theoretical calculations. Major part of the writing.
PAPER V: Part of experiments and calculations. Major part of writing.
vi
Contents
Abstract i
Sammanfattning ii
List of Papers iii
Author’s contribution v
List of Abbreviations viii
Declaration ix
1. Introduction ……………………………………..……………………………………………………..1
1.1. Chirality in life chemistry……………………………………………………………………1
1.2. Chiral recognition of ions in gas phase…....…………………………………………..3
1.3. Mass spectrometry based methods of chiral recognition………………………..4
1.3.1. Kinetic method……………………………………………………………………….5
1.3.2. Collision-induced dissociation of diastereomeric adducts…………..6
1.3.3. Host-guest diastereomeric adduct formation……………………………7
1.3.4. Ion-molecule reactions…………………………………………………………..7
1.3.5. Ion mobility spectrometry……………………………………………………...8
1.4. Infrared spectroscopy of chiral molecules…………………………………………...9
1.5. Aim of thesis…………………………………………………………………………………....10
2. Experimental and theoretical methods…………………………………………………11
2.1. Electrospray ionization (ESI)……………………………………………….…………...11
2.2. Experimental setup…………………………………………………………………………..12
2.2.1. ESI platform………………………………………………………………………….12
2.2.2. Time-of-flight mass spectrometry……………………………………………13
2.2.3. Fourier transform ion cyclotron resonance mass spectrometry….15
2.3. Theoretical methods…………………………………………………………………….…..17
2.3.1. Density functional theory (DFT)……… …………………………………....17
2.3.2. Self-consistent charge density functional tight binding
(SCC-DFTB) molecular dynamics (MD)………………………….………..17
3. Summary of results……………………………………………….………………………………..19
3.1. High-energy collisions of protonated enantiopure amino acids with a chiral
target gas………………………………………………………………………………………………19
vii
3.2. Low-energy collisions of protonated enantiopure amino acids with chiral
target gases……………………………………………………………………………………..….25
3.3. Chirally sensitive collision induced dissociation of proton-bound
diastereomeric complexes of tryptophan and 2-butanol……………….…………28
3.4. Non-covalently bonded diastereomeric adducts of amino acids and (S)-1-
phenylethanol in low-energy dissociative collisions…………………………….….32
3.5. IRMPD spectroscopy of protonated tryptophan diastereomers …………..35
4. Conclusions and outlook………………………………………………………………..42
Acknowledgements…………………………………………………………………………………..44
References……………………………………………………………………………………………….46
viii
List of Abbreviations
Ar Argon
CID Collision-induced dissociation
CoM Center-of-mass
DC Direct current
DFT Density functional theory
DFTB Density functional tight binding
DNA Deoxyribonucleic acid
ECD Electron-capture dissociation
ee Enantiomeric excess
ESI Electrospray ionization
FEL Free electron laser
FT-ICR Fourier transform ion cyclotron resonance
IMS Ion mobility spectrometry
IR Infrared
IRMPD Infrared resonant multiple photon dissociation
MCP Multi-channel plate
MD Molecular dynamics
Met Methionine
MS Mass spectrometry
MS/MS Tandem mass spectrometry
OPO Optical parametric oscillator
PC Personal computer
Phe Phenylalanine
Q-TOF Quadrupole time-of-flight
RF Radio frequency
SCC-DFTB Self-consistent charge density functional tight binding
TOF Time-of-flight
Trp Tryptophan
ix
Declaration
The work is partly taken from my licentiate thesis.
Chapter 1: sections 1.1, 1.2 (1.2.1, 1.2.2);
Chapter 2: sections 2.1, 2.2 (1.2.1, 2.2.2);
Chapter 3: sections 3.1, 3.3 have been partly modified and included in this thesis.
1
1. Introduction
1.1 Chirality in life chemistry
Chirality is a natural phenomenon that might seem simple at first sight, but on
closer inspection raises fundamental questions related to symmetry breaking and the origin
of life. The objects that have a particular handedness, left or right, are called chiral. Chiral
objects are non-superimposable mirror images of each other. Many examples of chirality
can be found around us, the large-scale as buildings, plants, seashells, our hands, the small-
scale, such as proteins, DNA and amino acids (Figure 1.1).
Figure 1.1: Examples of chiral structures made by human and nature (from left to right:
building; plant; seashell; protein alpha helix; DNA).
In living objects, usually one handedness is preferred. Either left-handed or right-
handed structures are present, but not both at the same time. This is called homo-chirality.
Even in the human body that looks symmetric, we can identify definite difference in
functions of symmetric parts, the preferential usage of one hand or the different functions
of the left and right half of our brain, etc. One would not expect much difference between
two structures that are as similar as the original and its mirror image, yet in nature this
minor inequality plays a critical role. The functioning of biological polymers and their
specificity in reactions are strongly dependent on the chirality of their constituents [1; 2].
In nature amino acids are present in left-handed form, and right-handed form is inherent
in carbohydrates. It has been generally accepted that life could not exist without molecular
dissymmetry and might be even a precondition [3; 4].
The first report of molecular chirality was made by Louis Pasteur in the middle of
19th century. In his experiments with tartrates, he explained the effect of polarized light
rotation while passing the light through the natural crystals of tartaric acid. Connecting
2
chemical, physical and crystallographic data, he established the definition of molecular
chirality [5 - 7]. Molecular structural isomers that are non-superimposable mirror images
of each other, are chiral and called enantiomers (from Greek “enántios” - opposite, and
“méros” - part) (Figure 1.2). An equal mixture of left and right enantiomers is called a
racemic mixture.
Figure 1.2: Schematic representation of two enantiomers of an amino acid (R is a side-
chain group).
Chiral molecules play a key role at all levels of biology, from bacteria to humans.
Most biological polymers consist of monomers having the same handedness, i.e., they are
homo-chiral. Several scenarios have been proposed with the aim at solving the question of
homochirality in life and its origin [3; 8 - 12]. These models propose various physical and
chemical phenomena to be responsible for symmetry breaking, and have been tested, yet
they remain speculative and none have been generally accepted [13].
Except for the ability to rotate plane-polarized light by equal amount in opposite
directions (optical isomerism), the physical properties of two enantiomers in a symmetric
environment are identical. However, in an asymmetric surrounding, their activity can be
substantially different. This is especially noticeable in living organisms. Due to their
chirality, left and right enantiomers of a compound can produce different physiological and
pharmacological effects [14 - 18]. Treatment of people with drugs consisting of a racemic
mixture without checking the effects of both enantiomers separately have already led to
several tragedies in human history [19]. For these safety reasons, together with the
superiority in efficiency of enantiopure drugs, enantiopure drug production, purity analysis
and separation of enantiomers is required. Nowadays, enantiopure drugs dominate among
all newly approved medication in the pharmaceutical industry [20; 21]. The research on
chiral molecules, their properties, and the development of new separation methods and
analysis are of great importance. Besides the needs for the pharmaceutical industry and
3
applications, the underlying mechanism behind symmetry breaking in molecules is a
fundamental question that has to be answered.
1.2 Chiral recognition of ions in gas phase
In order to characterise a mixture of chiral molecules, one usually has to determine
its enantiomeric composition in terms of enantiomeric excess (ee). If the amount of each
enantiomer in a mixture is known the ee can be determined as:
𝑒𝑒 =𝑅−𝑆
𝑅+𝑆 , (1.1)
where R and S are the fraction of the right- and left-handed enantiomers in the mixture,
respectively. The R and S notation refers to the Latin words “rectus” - strait/proper, and
“sinister” - left, and are defined by the spatial arrangement of the atoms in a chiral molecule
or by the Cahn–Ingold–Prelog priority rules [22]. Another notation system uses L or (-) and
D or (+) prefix (from Latin “laevus” - left and “dexter” - right) based on the direction in
which enantiomer rotates the plane of polarized light.
Several methods have been developed for chiral analysis including measurements
of vibrational optical activity [23; 24], optical rotatory dispersion [25], circular dichroism
[26], X-ray crystallography [27], Coulumb explosion imaging [28], microwave three-wave
mixing [28], nuclear magnetic resonance spectroscopy [29; 30], chromatography [30; 31],
and mass spectrometry (MS) based methods [32]. The latter one has unique advantages due
to its sensitivity and speed of analysis [33].
MS based methods provide an opportunity to study molecules in the gas phase, a
solvent-free environment without any interference of the surroundings. The direct
discrimination of enantiomers by MS is not possible since the masses of two enantiomers
are equal. In order to achieve separation, one should form diastereomeric (stereoisomers
that are non-mirror images of each other) complexes with a chiral selector, a compound
that interacts stereoselectively with the enantiomers of the substance of interest (analyte)
[34]. This is the crucial step in most gas phase chiral recognition techniques. The
interactions responsible for chiral recognition are different from case to case, including van
der Waals and hydrogen bonding, dipole–dipole and hydrophobic interactions.
Nevertheless, there are several similarities, which were generalized by Pirkle in a generally
accepted “three-point-model” or “three-point rule” [35]. The main idea of this model is
based on the requirement to have at least three points of interaction between the analyte
and the chiral selector [36]. In order to resolve two enantiomers, it is required that at least
three sites in the molecule of the chiral selector interact simultaneously with definite sites
4
of at least one of the enantiomers, and that one of these interactions must be stereo-
dependent [37]. The exchange of the enantiomers leads to a difference in the interaction
between the molecules (Figure 1.3), and a consequent change in the free energy of the
complex. Therefore, the separation of enantiomers becomes possible due to the
thermodynamic enantioselectivity [35; 38]. The three-point model has been widely used to
explain different enantiomeric separation techniques such as chiral liquid chromatography
[39] and mass spectrometry based methods [40]. However, the fundamental mechanisms
of chiral recognition are still under debate [36].
Figure 1.3: Schematic representation of the interaction sites of two enantiomers (top) with
a chiral selector (bottom).
1.3 Mass spectrometry based methods of chiral recognition
MS is a powerful tool in the field of biomolecular analysis in pharmaceutical and
biological sciences. MS based methods, with their speed of performance, have become a
potent technique of enantiomer analysis. The first successful chiral discrimination utilizing
MS was reported by Fales and Wright [41], and has undergone drastic development since
then. To date, several MS based methods for enantiomeric recognition have been developed.
Usually, they require ion-molecule reactions between the analyte and the chiral selector, and
can be divided into five specific groups [33]: (1) the kinetic method, (2) collision-induced
dissociation of diastereomeric adducts, (3) host-guest diastereomeric adduct formation, (4)
ion-molecule reactions and (5) ion mobility spectrometry. In the following section the main
concepts in each these groups are described.
5
1.3.1 Kinetic method
The kinetic method is based on the analysis of competitive ion cluster fragmentation
reactions. The proton-bound ionic complex is mass selected and exposed to collision-
induced dissociation (CID) in an experiment that involves multiple steps of MS (tandem
MS or MS/MS). The fraction of the fragment ionic species varies with the analyte chirality
[42 - 51]. The reaction rate constants, k1 and k2, that represent the particular dissociation
reaction channels, relate logarithmically to the gas-phase basicities of the complex
constituents B1 and B2:
𝑙𝑛(𝑘1
𝑘2) =
𝛥(𝛥𝐺)
𝑅𝑇𝑒𝑓𝑓, (1.2)
where Δ(ΔG) stands for the difference in the gas-phase basicities, R is the ideal gas constant,
and Teff is the effective temperature, the parameter that describes the average internal
energy of the activated ions [52]. Via the competitive dissociation in the ionic complex, one
can determine the value of Δ(ΔG). This is the basis of the kinetic method of chiral
recognition. The small energy difference (Δ(ΔG) < 1 kJ/mol) for dissociation leads to large
changes in the respective rate constant, which correlates with the relative abundance of the
corresponding fragments in the tandem MS experiment. By using the macromolecular
complexes formed by three, usually non-covalently bound, entities (trimeric complexes),
more sterical effects can be involved, which consequently leads to magnification of Δ(ΔG)
and improves the enantioselectivity. This assumes the following reaction:
where An is an analyte, ref* is a reference ligand, M is a metal ion. The schematic
representation of the reaction energy is shown in Figure 1.4.
6
Figure 1.4: The diagram of competing reactions in the kinetic method for a chiral analysis
using trimeric complexes [33].
1.3.2 Collision-induced dissociation of diastereomeric adducts
Diastereomeric adducts that differ by the substitution of the enantiomer have
different stability due to the various interaction sites between its constituents. In this
method, preformed diastereomeric adducts are mass selected and exposed to collisions with
a neutral gas. The energy transferred in collisions is redistributed among the available
internal modes and leads to the fragmentation of the adduct. The resulting relative
abundance of the fragments and dissociation pathways are determined, and the data for the
two enantiomers compared [53 - 55] (Figure 1.5).
Figure 1.5: Scheme of chiral recognition via collision-induced dissociation of
diastereomeric adducts [56].
7
1.3.3 Host–guest diastereomeric adduct formation
The method uses single stage MS and isotopically labeled enantiomer compounds
in order to monitor the formation of the host-guest diastereomeric adducts by analysing
their abundances. Since one enantiomer is isotopically labeled they can be resolved in the
mass spectrum [57 - 71] (Figure 1.6).
Figure 1.6: Scheme of guest–host diastereomeric adduct formation for chiral recognition
[33].
The degree of chiral discrimination is determined by the ratio of peak intensities IR and IS
and called IRIS.
𝐼[(𝐻𝑅 + 𝐺)+]
𝐼[(𝐻𝑆−𝑖.𝑙. + 𝐺)+]=
𝐼𝑅
𝐼𝑆−𝑖.𝑙.= 𝐼𝑅𝐼𝑆 (1.3)
1.3.4 Ion-molecule reactions
In this method of chiral recognition the host-guest ion-molecular complexes are
formed, mass selected and trapped in an ion trap with a neutral reagent. The guest-
exchange reaction takes place, which is strongly dependent on the chirality of the guest
molecule (Figure 1.7). The enantioselectivity is determined by the ratio γ of the reaction rate
constants kR and kS for each enantiomer [72 - 79].
Figure 1.7: Scheme of equilibrium host–guest complexes reactions [33].
8
𝛾 = 𝑘𝑅𝑘𝑆
(1.4)
By comparison of the relative abundance of the reaction products with known values of ee,
the calibration curves are constructed, which is used for the chiral analysis.
1.3.5 Ion mobility spectrometry
Ion mobility spectrometry is a very promising technique for stereoisomerism
investigations [80]. The discrimination is dependent on the collision cross section of the
molecules and their charge. The ion mobility setup consists of an ion source, a drift tube,
and a detector (Figure 1.8). The ionised analyte is transferred to the drift tube by means of
applied electric field. The ions move in the drift tube and undergo multiple collisions with
the drift gas. Depending on the charge and the collision cross section they migrate at a
different rate and hence are detected at a different time.
Figure 1.8: Principle scheme of ion mobility spectrometry setup.
The drift velocity, vD, of the ions is proportional to the magnitude of the electric field
strength, E, and the ion cloud mobility K [81].
𝑣𝑑 = 𝐾𝐸 (1.5)
The mobility of the ion cloud, K, is a function of the ion charge, q, temperature, T, reduced
mass of the ion and the drift gas molecules, μ, the number density of the drift gas, N, and
the ion-molecule collision cross section, ΩD:
9
𝐾 = (3𝑞
16𝑁)(
2𝜋
𝜇𝑘𝑇)
1
21
𝛺𝐷 (1.6)
Enantiomeric separation is achieved by adding a chiral dopant to the drift gas. This can
result in the stereo-dependent long-range interaction between ions of chiral analyte and
chiral dopant, and lead to variation of the drift time for different enantiomers [82; 83].
The first chiral separation by means of ion mobility spectrometry was demonstrated by
Dwivedi in 2006 on enantiomers of different amino acids using S-2-butanol as a chiral
dopant added to the drift gas. The mechanism was explained based on the Pirkle three-
point model interaction [35]. Since then, the technique has been developed by number of
groups [84 - 87], yet Dwivedi’s results have not been reproduced. The chiral IMS
mechanism via short-lived cluster formation has been proposed. This mechanism can also
explain the enantiomer separation in IMS using the achiral modifier [88; 89]. However a
full understanding of the chiral IMS is still lacking.
1.4 Infrared spectroscopy of chiral molecules
Vibrational spectroscopy is a well-developed method for structural analysis. The
analyte is irradiated with an IR laser over a specific range of wavenumbers. The resonant
absorption of multiple IR photons results in the fragmentation of the species and
consequent detection of the dissociation reaction product. Combining the information
about the fragments, absorption efficiency and photon energy the IR spectra is obtained.
Infrared resonant multiple photon dissociation (IRMPD) spectroscopy coupled to mass
spectrometry gives the IR spectra and combined with the quantum chemical calculation
allows studying vibrational modes, perform structural and conformational analysis of the
ions and ionic clusters (Figure 1.9).
10
Figure 1.9: The structural analysis performed by comparison of experimental (black) and
calculated for three different conformers (blue A, B and Z) IR spectra.
Two enantiomers have identical IR spectra, therefore to make a differentiation of
enantiomers possible, the formation of diastereomers is needed. The first demonstration of
chiral recognition incorporating protonated diastereomeric complexes of organic molecules
has been done by Scuderi et. al. [90]. The difference in the structure of diastereomers, based
on different bonding and points of interaction, results in the difference in the IR spectrum.
1.5 Aim of thesis
The aim of this thesis is to deepen the understanding of chiral recognition mechanisms and
shed light on the main processes that lead to the discrimination of enantiomers. In order to
reveal the corresponding process, we try to simplify the experiments to target the
fundamental interactions. We deliberately avoided possible environmental effects, the
utilisation of metals for steric effect amplifications, multiple collisions and high degree of
clustering. We have studied the ion-molecule reactions in the gas phase: the interactions of
enantiopure amino acids in high- and low-energy collisions with achiral and chiral targets;
diastereomeric adduct formation and dissociation processes; resonance photoabsorption of
diastereomeric proton bound dimers.
11
2. Experimental and theoretical methods
The instruments used for the experiments presented in this work, utilised the
electrospray ionization (ESI) technique to produce ions. The main characteristics of the
experimental apparatuses, the ESI technique, and the theoretical methods used in this work
are described below.
2.1 Electrospray ionization
Electrospray ionization is a powerful technique for producing intact ions in vacuo
from species in solution [91]. Although it has been widely used for some time, the precise
mechanism of ESI is still under debate [92; 93]. One probable electrospray ionization
mechanism is shown on Figure 2.1.
Figure 2.1: Mechanism of electrospray ionization.
A solution that contains the substance of interest is dissolved in a solvent (usually water,
methanol or a mixture that also often contains an acid as a source of protons). Emerging
from the spraying nozzle, the liquid disperses into an aerosol due to the strong electric field
of about several kV at the needle tip. The jet forms at an electric field, which is higher than
a certain threshold value and strongly depends on the solution’s surface tension. Droplets,
guided by an electric field, pass through a heated capillary that connects to the main vacuum
system of the apparatus. The solvent evaporates from the droplets, resulting in their
shrinkage. Consequently, the surface charge density increases. The droplets burst when the
Coulomb repulsion becomes stronger than the surface tension. The smaller product
droplets follow the same procedure until the analyte is free of solvent molecules. This
12
particular mechanism describing the ESI process is called the charged-residue model, and
was proposed by Dole et al. [94]. Another possible mechanism is called the ion evaporation
model [95], where the protonated analyte ions are lifted from the nm-size droplets into the
gas phase by a strong electric field.
In both models there is lack of knowledge about the dispersion of solution into charged
droplets as well as the formation of a single analyte ion. This process is strongly dependent
on many variables, in particular the electric field strength, the solution flow rate, and
properties of the liquid (concentration, pH, dielectric constant, etc.). ESI is a method of soft
ionization, especially widespread in MS biomolecular analysis, since large fragile molecules,
such as proteins and enzymes, can be easily ionized and transferred to the gas phase without
their destruction [96].
2.2. Experimental setup
2.2.1 ESI platform
The study of keV energy collisions of protonated amino acids with chiral and achiral
targets was performed at the electrospray ion source platform, designed and constructed at
the Department of Physics, Stockholm University [97]. A scheme of the apparatus is shown
in Figure 2.2.
Figure 2.2: Sketch of the experimental setup used in the keV energy collisions experiment
[97].
The ions that come from the heated capillary are introduced to the ion funnel where they
are exposed to collisional focusing in co-applied RF and DC fields. The funnel was designed
and manufactured at Stockholm University [97]. The effective radially focusing potential
13
and the DC field result in efficient ion transfer through the system. Ions exhibit multiple
collisions with the residual gas, which enhance the focusing of the beam. The focusing
potential is created by means of an applied RF field. The instrument can operate in
continuous and pulsed mode due to the linear RF octupole, which follows the ion funnel,
and can work as an ion guide or accumulation trap. An octupole ion guide is located directly
after the RF octupole in order to transport ions through the differential pumping stage. The
ions then pass into a quadrupole mass filter. The possible mass-to-charge range is 2-4000
amu/q. RF filters are installed before and after the quadrupole to improve the ion
transmission. After the quadrupole mass filter a set of focusing and deflecting elements are
installed for the same reason. After mass selection, the ions are accelerated to the required
energy, and located after the acceleration stack in the 4 cm long collision cell. It can be filled
with gas in order to study collisions of ions with neutral targets. The analysis of the ions
generated in collisions is made by means of a cylinder lens and two pairs of electrostatic
deflector plates. A 40 mm double-stack multi-channel plate (MCP) with a position sensitive
resistive anode serves as a detector of the ionic fragments. The data are acquired and
processed with a PC and the specially developed software system.
2.2.2 Time-of-flight mass spectrometry
Many fundamental concepts of time-of-flight mass spectrometry (TOF MS) were
developed by Wiley and McLaren in 1950s [98]. Since then, this kind of mass spectrometry
has been greatly developed. Nevertheless, the main principles remain the same and can be
divided in three sequential steps: ion formation, acceleration, and separation in the drift
region. Figure 2.3 illustrates the main principles of TOF MS. The detailed description of
TOF MS can be found in many reviews and papers [99 - 102]. Here we describe the main
fundamentals of TOF MS that are essential for understanding of our experiments.
Figure 2.3: Principle scheme of time-of-flight mass spectrometer.
Ions of mass m and charge q are formed in bunches. Starting at defined time, they
are transferred by means of an electric field, E, through an accelerating region, sa, to the
14
drift region, D. In the electric field they gain a kinetic energy Ek. Ideally, the ions enter the
drift region with the same Ek.
𝐸𝑘 = 𝑞𝐸𝑠𝑎 =1
2𝑚𝑣2 (2.1)
Then the drift time can be written as:
𝑡𝐷 = 𝐷√𝑚
2𝑞𝐸𝑠𝑎 (2.2)
The velocity of the ions is inversely proportional to the square root of the mass-to-charge
ratio m/q. The ions drift a distance D, leading to a continuing separation from each other,
and reaching the detector at different drift times that depend on their mass-to-charge ratio.
The quadrupole time-of-flight mass spectrometer (Q-TOF) was used in the
experiments with an ESI source, see Figure 2.4. This apparatus could be separated into two
parts: the quadrupole mass analyzer and an orthogonal acceleration TOF mass
spectrometer. The hexapole collision cell makes it possible to run this apparatus in tandem
MS mode, which provides the additional structural information via fragmentation. Ions
produced by means of the electrospray source are transferred to the quadrupole via an RF
lens. Ion acceleration, focusing, steering and tube lenses shape and focus the beam into the
pusher. Then ions are introduced in pulses into the reflectron section and monitored by the
microchannel plate detector and counting system. The available operating frequencies of
the pusher can be up to 20 kHz, which results in the recording of the whole spectrum every
50 microseconds.
Figure 2.4: Diagram of Q-TOF mass spectrometer used in the experiment done at
University of Oslo [103].
15
The data are acquired and processed with a PC and the MassLynx NT software system
required to control the Q-TOF apparatus.
2.2.3 Fourier transform ion cyclotron resonance mass spectrometry
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is a
powerful technique for investigation of ions in the gas phase [104]. The ability to analyse
the complex molecular mixtures, ion-molecule reactions, their energy characteristics,
equilibria and kinetics, have made FT-ICR MS widely used especially in chemical physics,
proteomics and metabolomics [105]. The principle elements of FT-ICR MS is the ion source,
magnet, ion trap, ion optics, and the system of ion excitation and detection Figure 2.5.
Figure 2.5: Sketch of the IR spectroscopy experimental setup utilized for the infrared
resonant multiple photon dissociation (IRMPD) experiment in Orsay, France.
Superconducting magnets are used to obtain strong magnetic field, which is a
determining factor for the resolving power, mass accuracy, mass and dynamic range of the
mass spectrometer [106]. A combination of the electric and strong magnetic fields traps the
ions in the ion cyclotron resonance cell. They rotate with arbitrary phases in the plane
perpendicular to the magnetic field. A radio frequency voltage is applied to the excitation
electrodes to induce a synchronous cyclotron motion of the ions. The detection is done by
recording the induced image charge on the trap detection electrodes (Figure 2.6).
16
Figure 2.6: Principle scheme of ICR mass spectrometry.
The potential changes on these electrodes is processed using a Fourier transform.
The mass to charge ratio is derived from the frequency analysis. This frequency, wc, is
defined by the magnetic field, B, the charge, q, and mass, m, of the ion.
𝑤𝑐 =𝑞
𝑚𝐵 (2.3)
To study particular reactions different types of ion excitation are used in FT-ICR
experiments. The most common processes that are utilized for the bio-molecular analysis
are electron-capture dissociation (ECD) [107], CID and IRMPD.
17
2.3 Theoretical methods
2.3.1 Density functional theory (DFT)
Density functional theory is a computational quantum mechanical method. Because of its
computational efficiency and relatively good accuracy it is widely used for investigating the
electronic structure of molecules in quantum chemistry, chemical physics, solid-state
physics, etc. It is based on the description of many-electron system with the electron density
functional. The exact knowledge of the wave function is not needed. The electronic energy
of the ground state is determined by the electron density, ρ(r) [108]:
𝐸𝐷𝐹𝑇[𝜌] = ∫ 𝑈𝑒𝑥𝑡(𝑟)𝜌(𝑟)𝑑𝑟 + 𝐹[𝜌], (2.4)
where EDFT[ρ] is the functional of a particular electron density, ρ. The external potential,
Uext(r), is system-dependent potential, specific for each case. Independent of the Uext(r)
functional, F[ρ], is represented as a sum of T[ρ] and V[ρ], the electronic kinetic and
potential energy functionals respectively:
𝐹[𝜌] = 𝑇[𝜌] + 𝑉[𝜌]. (2.5)
The electron interaction is represented using the Kohn-Sham DFT [109]. It treats
interaction as a set of non-interacting electrons in the effective potential, which result in the
same electron density as for interacting electrons, and has the following functional form:
𝐹[𝜌] = 𝑇𝐾𝑆[𝜌] + 𝐽[𝜌] + 𝐸𝑋𝐶[𝜌], (2.6)
where TKS[ρ] is a Kohn-Sham kinetic energy functional; J[ρ] is a functional of the Coulomb
energy; EXC[ρ] is a functional of the exchange correlation energy. TKS[ρ] represents the
kinetic energy of non-interacting electrons. The determination of the electron density is
done in a self-consistent manner. The Kohn-Sham DFT energy functional is defined as
𝐸𝐾𝑆[𝜌] = ∫ 𝑈𝑒𝑥𝑡(𝑟)𝜌(𝑟)𝑑𝑟 + 𝑇𝐾𝑆[𝜌] + 𝐽[𝜌] + 𝐸𝑋𝐶[𝜌]. (2.7)
The exact exchange correlation functional is unknown and can be only derived in
approximate form.
2.3.2 Self-consistent charge density functional tight binding (SCC-DFTB) molecular
dynamics (MD)
SCC-DFTB is an approximate quantum chemical method. It is based on the
simplified DFT algorithm that reduces the computational costs, and can serve as a good
alternative to classical force field methods. It is comparable to semi-empirical methods such
as AM1 [110] and PM3 [111; 112]. Yet, no explicitly empirical values are used in the method
18
for the parameterization. In some cases the accuracy of the calculations performed with
SCC-DFTB can be comparable to DFT calculations [113; 114]. The method is based on the
second-order expansion of the exchange-correlation functional with respect to the electron
density [115]. There are several simplifications applied in the method to reduce the
computational costs: the minimum basis set of atomic orbitals is used; the calculations are
performed for the two-center interactions. The tabulated values from DFT calculations for
overlap of the atomic orbitals in diatomic interaction are utilized for this purpose. The
contribution of the partial charge of each atom to the total energy is calculated in a self-
consistent way in the process of the system optimisation [115]. SCC-DFTB method was used
in this work for MD simulations. MD was the first step of optimisation and conformational
analysis in the papers III, IV and V. Obtained with DFTB+ software [116] structures were
used as an input geometry for further optimisation with DFT methods.
19
3. Summary of results
3.1 High-energy collisions of protonated enantiopure amino acids with
a chiral target gas
In this work we have studied collision-induced dissociation of singly protonated
enantiopure amino acids, namely tryptophan (Trp), phenylalanine (Phe) and methionine
(Met) with respect to the nature of the target gas. Collisions of 1 keV in the center-of-mass
(CoM) frame with argon, (±)-2-butanol and S-(+)-2-butanol as the targets were performed.
The chemical structure of the molecules used in the experiments are shown in Figure 3.1.
Figure 3.1: Chemical structure of methionine, phenylalanine, tryptophan, and 2-butanol.
The chiral centers are marked with the red circle.
The resulting CID mass spectra were analysed and explained with the generally
accepted concepts used to explain the mechanism of protonated amino acid dissociation:
the mobile proton model [117; 118], and a model that involves the side-chain group in a
dissociative reaction [118; 119] (Scheme 1).
Scheme 1. Scheme of main dissociation pathway of protonated amino acids (R - side-chain
group).
20
Figure 3.2: Mass spectrum obtained from collisions of PheH+ with (a) argon; (b) racemic
(±)-2-butanol; (c) (S)-(+)-2-butanol.
Figure 3.2 collects the CID spectra obtained from collisions of protonated PheH+
with argon (Fig. 3.2a), racemic (±)-2-butanol (Fig. 3.2b), and (S)-(+)-2-butanol (Fig. 3.2c).
CID of PheH+ mainly goes via the loss of H2O and CO with consequent formation of the
iminium fragment at m/z = 120 (Scheme 1, Pathway A), which is in good agreement with
previously reported results [120 - 123]. Formation of the iminium ion is a typical amino acid
dissociation pathway. Several additional fragments were observed in the mass spectrum,
which indicates the presence of competing dissociation channels. The collision energy of 1
keV is sufficient to initiate several fragmentation channels. The competing reaction is the
loss of NH3 (Scheme 1, Pathway B). Further fragmentation of the ion, following loss of NH3,
leads to formation of fragments with m/z = 131 (due to loss of H2O) and m/z = 103 (due to
loss of H2O + CO). The mass spectra obtained with different enantiomers of PheH+ have
been compared. The difference in the relative abundance of the generated fragments was
21
found to be not statistically significant. The reaction channels were the same for each
enantiomeric form of amino acids. For chiral and achiral targets in the CID of PheH+, no
stereo-dependent effects were observed.
Figure 3.3 collects the CID spectra obtained from collisions of TrpH+ with: argon
(Fig. 3.3a), racemic (±)-2-butanol (Fig. 3.3b) and (S)-(+)-2-butanol (Fig. 3.3c). The main
fragmentation channel in CID of TrpH+ was the loss of NH3 (m/z = 188) (Figure 3.3a;
Scheme 1, Pathway B), which is different from the generally accepted dissociation pathway
via combined loss of H2O and CO for other aromatic amino acids. However, the [CO + H2O
loss] fragment (m/z = 159) was also detected (Figure 3.3a; Scheme 1, Pathway A), about 5
times less abundant than m/z = 188. Formation of an ion at m/z = 159 is in a good
agreement with the proton transfer model reported earlier [124 - 127], yet it is in contrast
to the report of Rogalewicz et al. [128], who proposed a theoretical explanation of TrpH+
fragmentation without production of the iminium ion in the dissociation process. The
collision energy of 1 keV was sufficient to initiate further thermally driven dissociation with
the main fragments formed due to loss of CH2CO (m/z = 146), CO2 + H• (m/z = 144), and
CH2CO + CO (m/z = 118) (Figure 3.3).
In order to investigate the influence of the target on the fragmentation channels and
the fragment abundances, we compared the mass spectra generated in the CID of S- and R-
TrpH+ with argon, racemic and (S)-(+)-2-butanol. We found that the generated CID
fragments and their relative abundance were independent of the target nature with only two
exceptions; at m/z = 130 and m/z = 188, which changed dramatically with the target, but
not with the different forms of TrpH+. This might be due to differences in electrospray
conditions during the experiments, which resulted in different internal energy of the
generated ions. Another possible explanation is the presence of the Trp radical cation in the
ion beam, generated during beam focusing in the ion funnel. The m/z = 130 fragment is not
a characteristic one for TrpH+ but is a fingerprint of the Trp cation radical fragmentation
[129]. The available resolution did not allow us to resolve m/z = 205 and m/z = 204.
22
Figure 3.3: Mass spectrum of TrpH+ obtained in collisions with (a) argon; (b) racemic (±)-
2-butanol; (c) (S)-(+)-2-butanol.
Figure 3.4 collects the CID spectra obtained from collisions of MetH+ with argon
(Fig. 3.4a), racemic (±)-2-butanol (Fig. 3.4b) and (S)-(+)-2-butanol (Fig. 3.4c). 1 keV energy
collisions of protonated MetH+ with Ar, racemic 2-butanol and (S)-(+)-2-butanol results in
loss of NH3 (m/z = 133) (Figure 3.4; Scheme 1, Pathway B) and H2O + CO (m/z = 104,
Scheme 1, Pathway A). Peaks at m/z = 115 correspond to successive loss of NH3 and H2O.
The peak at m/z = 102 corresponds to side chain loss of CH3SH. Unfortunately, the
resolution limitation restricted us from resolving this peak from the peak at m/z = 103,
which is due to loss of H2O and CO.
23
Figure 3.4: Mass spectra of MetH+ after collisions with (a) argon; (b) racemic (±)-2-
butanol; (c) (S)-(+)-2-butanol.
Again, comparison of the mass spectra obtained in experiments with enantiomers
of MetH+ with different target gases was made. The fragmentation pattern and relative
abundance of the fragments showed no differences. However, the different gases showed
an influence on the dissociation pathway, which most likely can be explained as in the case
of the TrpH+, that is by the difference in electrospray conditions. A fragment at m/z = 61 is
the most probable product of the Met cation radical dissociation [130].
In 1 keV energy collisions of protonated enantiomers of amino acids with neutral
chiral and achiral targets we did not detect any stereo effects. The amount of energy gained
in the collisions was sufficiently high to generate abundant fragmentation with several
competing dissociation reactions. The loss of H2O + CO and NH3 were observed for each
amino acid used in the experiments. Our results are in agreement with the models of mobile
24
proton and participation of side chain groups [117 - 119]. The statistical character of the
fragmentation of the amino acids can be explained by the dominance of electronic
excitation. No chiral effects were observed in our work. It is likely that the energy of 1 keV
is too high for stereo-chemical effects to occur. Observed differences of fragmentation in 1
keV experiment were most likely due to minor changes in electrospray source parameters.
25
3.2 Low-energy collisions of protonated enantiopure amino acids
with chiral target gases
In the 1 keV collisions of the enantiopure amino acids with chiral and achiral targets,
the collision energy was too high to detect possible stereo-dependent interaction of the
projectile and the target [131]. In this study we focused on the low-energy collisions of
enantiopure protonated amino acids (PheH+, MetH+ and TrpH+) with chiral targets (2-
butanol and 1-phenylethanol). The experiments were done in a single collision mode. Two
processes have been observed in these experiments: the fragmentation of the amino acids
and the formation of a proton bound adduct consisted of the protonated amino acid and the
neutral target gas. Similarly to the high-energy collisions, the fragmentation of the
protonated amino acids goes via generally accepted schemes: PheH+ loses H2O and CO
resulting in a protonated fragment at m/z = 120 (mobile proton model [131; 117; 118]);
TrpH+ fragmentation goes via the sidechain-group participation [131; 117; 118] resulting in
loss of NH3 (fragment at m/z = 188). For MetH+ we observed both fragmentation channels
and corresponding products at m/z = 133, the loss of NH3 group, and at m/z = 104, the
combined loss of H2O and CO. In comparison to the high-energy experiments [131], the low-
energy collisions led to much lower yields of fragments. We utilized both enantiomeric
forms of the amino acids and target gases. The fragmentation was independent of the
chirality of studied molecules. Additional experiments with racemic mixture of (±)-2-
butanol as a target gas confirmed our findings. The formation of a collision complex
consisting of the protonated projectile and the target was the main reaction channel at
energies lower than 0.6 eV lab frame for all amino acids used in the experiments (Figure
3.5).
26
Figure 3.5: Mass spectra obtained from collisions of (a and d) S/R-MetH+ (b and e) S/R-
PheH+ (c and f) S/R-TrpH+ with (S)-2-butanol (top) and (R)-2-butanol (bottom) at 0.1 eV
lab-frame energy.
The reaction was studied as a function of collision energy and investigated for
enantioselectivity by substitution of the projectiles and targets by their enantiomers. The
analysis of the stereo-dependence was done by comparing the relative abundance of the
collision complex formed in the reaction with (S)- and (R)-2-butanol (Figure 3.6).
Figure 3.6: Relative abundance of S- and R- TrpH+/PheH+/MetH++ (a) (S)-2-butanol; (b)
(R)-2-butanol adduct. Data is shown as a function of CoM collision energy.
27
As can be observed from Figure 3.6, complex formation is independent of the
chirality of the amino acids utilised in this experiment at all energies. Furthermore,
changing the collision gas to the other chiral form leads to the same result. We also
undertook similar experiment with the bulkier (S)-1-phenylethanol as a collision target.
The formation of complex was observed at the energy range 0.03 - 0.45 eV CoM frame
(Figure 3.7). Both chiral forms of the amino acids have been utilized. The results of the
experiment are represented in Figure 3.7. Again the complex formation reaction showed no
stereoselectivity.
Figure 3.7: Relative abundance of S- and R- TrpH+/PheH+/MetH++ (S)-1-phenylethanol
adduct. Data is shown as a function of CoM collision energy. The parent peak is set to 1000.
The efficiency of both reaction channels, the fragmentation and the adduct
formation, was independent of stereo-chemical composition of the reactants. The result
suggested that the three-point model [35], used to explain the chiral discrimination of
amino acid enantiomers with 2-butanol as a chiral modifier in IMS [83] is unlikely to be
applicable to the ion-molecule interactions in our experiment. These results can also
indirectly support the chiral discrimination mechanism via clustering proposed by Holness
et. al. [88; 89].
28
3.3 Chirally sensitive collision induced dissociation of proton-bound
diastereomeric complexes of tryptophan and 2-butanol
In this study we modified the low-energy collision experiments by forming the
diastereomeric adducts in the electrospray source. Preformed adduct of TrpH+ and 2-
butanol was exposed to CID with argon (Ar) at low-energy collisions in a single collision
mode. The dissociation reaction (Scheme 2) was examined for enantioselectivity.
TrpH+-2-butanol + Ar → TrpH+ + 2-butanol + Ar
Scheme 2. Studied dissociation reaction.
The resulting mass spectrum is shown in Figure 3.8.
Figure 3.8: Mass spectrum obtained in CID of the TrpH+-2-butanol adduct.
The proton-bound adduct (m/z = 279) easily dissociates to its constituents, TrpH+
(m/z = 205) and neutral 2-butanol (Figure 3.8). We tested both enantiomeric forms of the
amino acid and 2-butanol in our experiment, and investigated the efficiency of the
dissociation reaction as a function of collision energy. The obtained mass spectra, in
particular the bond breaking channel, was analysed for stereo-effects as shown in Figure
3.9.
29
Figure 3.9: Abundance of S- and R-TrpH+ fragments gained in CID of the S- and R-TrpH+-
(a) (S)-2-butanol; (b) (R)-2-butanol, adduct as a function of CoM collision energy. The
parent peak is set to 1000.
The homo-chiral adduct appears to be less stable towards CID than the hetero-chiral
counterpart. The difference in the complexes that were used in the experiment was only in
the chirality of Trp. Therefore we assume that the dissociation efficiency depends on the
interaction between the complex constituents. Depending on the chirality the multiple
interaction points are different, which results in a stereo-dependent dissociation. This
finding was confirmed by additional experiments performed using (R)-2-butanol. All
experimental parameters were kept the same. Again, the hetero-chiral complex was found
to be more stable. The homo-chiral complex generates a more abundant peak
corresponding to TrpH+ and is therefore, less stable, see Figure 3.9b.
Additional experiments were performed to measure the CID of enantiomeric
complexes as a function of target gas pressure. The collision energy was kept at 4 eV (lab-
frame) and the gas cell pressure was varied in the range of 0.25 - 9.98 x 10-4 mbar. The
intensity of the intermolecular bond breaking fragment was investigated, and these data are
plotted in Figure 3.10. As we have observed in our previous experiments, the homo-chiral
complex fragments more efficiently.
30
Figure 3.10: Relative abundance of (S)- and (R)-TrpH+ fragments acquired in the CID of
the proton bound complex of (S)- and (R)-TrpH+-(S)-2-butanol with argon as a function of
the target gas pressure. The parent peak is set to 1000.
This finding confirms the stereo-dependent interaction between the molecules in
the above mentioned complex, which leads to chirality dependent dissociation. Our
experimental results provide strong evidence for the stereo-dependent dissociation of
TrpH+-2-butanol diastereomeric complexes.
To confirm the experimental results, quantum chemical calculations have been
carried out. First, molecular dynamics have been performed using the density functional-
based tight binding method (DFTB) [116]. In this first round of calculations based on the
electronic energy of the adduct, the structures of the most stable conformers were found.
Two types of bonding were observed in the MD simulations. The most stable type of
bonding was the intermolecular bond between the alcohol hydroxyl group and the
protonated amino group of amino acid. The hydrogen bond between the hydroxyl group of
2-butanol and the nitrogen on the Trp indole ring was found to be less stable and was not
included in further optimizations. The second round of optimization was done using the
Gaussian 09 package [132]. The most stable structures obtained after MD was optimized at
the B3LYP/6-31+G** level of theory (Figure 3.11).
31
Figure 3.11: Structures of the most stable conformers optimized with Gaussian 09 at
B3LYP/6-31+G** level of theory for (a) the homo-chiral S-TrpH+-(S)-2-butanol adduct; (b)
the hetero-chiral R-Trp-H+-(S)-2-butanol adduct.
(S)-2-butanol in the hetero-chiral adduct is located in a close proximity to the aromatic ring
of TrpH+ (Figure 3.11b). This orientation can induce stereospecific interactions, which is
not possible with the homo-chiral adduct as seen in Figure 3.11a. In the latter, (S)-2-butanol
is far from the aromatic rings of Trp, something which limits their interaction. In addition,
the intramolecular bond is formed in the homo-chiral adduct between the carboxyl group
and the nitrogen on the indole ring of TrpH+. Such bonding can play an additional
stereoselective role in the reaction. The electronic energy difference between the homo- and
hetero-chiral complexes was compared. Hetero-chiral R-TrpH+-(S)-2-butanol is 61 meV
collisions energy resolution lower than homo-chiral R-TrpH+-(S)-2-butanol, and confirms
our experimental results. However, calculations with the (R)-2-butanol as a chiral selector
gave the opposite result, with the hetero-chiral S-TrpH+-(R)-2-butanol adduct results 28
meV higher in energy than the homo-chiral adduct. This controversial result requires
further investigation, with more conformers included and different theoretical methods
utilized for the calculations. Additionally, the dispersion forces should be included in
calculations.
32
3.4 Non-covalently bonded diastereomeric adducts of amino acids and
(S)-1-phenylethanol in low-energy dissociative collisions
The stereo-dependent CID of diastereomeric adducts of TrpH+-2-butanol observed in our
previous work led us to a similar experiment with a different chiral selector. Using the same
three amino acids, bulkier (S)-(-)-1-phenylethanol was chosen as a selector in order to
amplify the stereo-dependent effect, observed in the dissociation reaction with 2-butanol.
The single collision mode with Ar was utilized. The adducts were formed in the electrospray,
mass selected and exposed to the collisions with the argon at 0.5 eV CoM energy. The
reaction proceeded via breaking the proton bond, producing protonated amino acid and
neutral alcohol as fragments (Figure 3.12). The energy used in the experiment was
insufficient to produce further fragmentation.
Figure 3.12: Mass spectra of (a) S/R-TrpH+-(S)-(-)-1-phenylethanol, (b) S/R-PheH+-(S)-
(-)-1-phenylethanol, (c) S/R-MetH+-(S)-(-)-1-phenylethanol adducts obtained in collisions
with Ar at 0.5 eV CoM energy.
In order to study the chiral dependence of the reaction, both enantiomers of amino acids
have been used. The resulting relative abundance of the fragments have been compared
(Figure 3.12). No difference was found in the fragment abundance for any of the
diastereomeric adducts. With this result, we conclude that there is no stereo-dependence of
the reaction using (S)-(-)-1-phenylethanol as a chiral selector. This is in contrast to the
definite chiral discrimination observed in CID of TrpH+-2-butanol [56].
In order to compare the results and analyse experimental data, quantum chemical
calculations were performed. The calculation procedure was similar to TrpH+-2-butanol in
33
our previous work. After MD simulation the most stable conformers within 3 kcal/mol
electronic energy difference from the most stable one have been taken for further DFT
optimization using Gaussian 09 [132] at B3LYP/6-31++G** level of theory. Additionally,
empirical dispersion has been taken into account in calculations. As a result, for each
diastereomeric adduct, the most populated (more than 95%, with the only exception for R-
PheH+-(S)-1-phenylethanol with 84% of population) conformation was identified. Each
adduct is formed via the hydrogen bond between the hydroxyl group and the primary amino
group in the amino acid, as illustrated in Figure 3.13, where the lowest energy conformer
has ΔG = 0.
Figure 3.13: Structures of the most stable conformers obtained after DFT optimization at
B3LYP/6-31++G** level of theory for homo- (left) and hetero-chiral (right) adducts of (a)
TrpH+-(S)-(-)-1-phenylethanol, (b) PheH+-(S)-(-)-1-phenylethanol, (c) MetH+-(S)-(-)-1-
phenylethanol. Figure from Paper IV.
The relative stability of the complexes has been compared in terms of their free
energy calculated at 298.15 K. As is shown in Figure 3.13, the hetero-chiral adducts are more
stable compared to the homo-chiral ones.
34
The structures of diastereomeric TrpH+-(S)-(-)-1-phenylethanol have different
orientations of the hydroxy group of Trp. In the homo-chiral adduct, it forms the
intramolecular hydrogen bond with the nitrogen in the indole ring. The free energy
difference is 4.38 kcal/mol. The homo-chiral diastereomer of S-PheH+-(S)-(-)-1-
phenylethanol has parallel orientation of the aromatic rings of alcohol and amino acid. In
contrast, hetero-chiral adduct have the antiparallel structure. The free energy difference is
5.36 kcal/mol, indicating higher stability for the hetero-chiral adduct at room temperature.
In the case of adducts with Met, the free energy difference is 1.21 kcal/mol, again favouring
the hetero-chiral adduct.
The use of phenylethanol as a chiral selector did not lead to any chiral discrimination
compared with 2-butanol in our experiments with TrpH+. The CID reaction turned out to
be chirally independent in this case. The amplification of the effect was not achieved using
the bulkier molecule as a modifier. The quantum calculations show the difference in the
structure and the free energy of the diastereomeric adducts for each amino acid, and
consequently show that the hetero-chiral adduct is more stable. Obtained in calculations
diastereomeric adducts have different structures and the unambiguous assignment of all
interaction points is essential. The temperature as a crucial parameter for conformers
population has to be monitored and controlled during the experiment.
35
3.5 IRMPD spectroscopy of protonated tryptophan diastereomers
In this work we investigated the structure and vibrational spectroscopy of proton
bound diastereomeric dimers of Trp employing a free electron laser (FEL) and a tabletop
OPO laser as the light sources. The measurements within the 2700-3750 cm-1 range were
undertaken for the homo-chiral LL-Trp2H+ and the racemic mixture of Trp. Additionally,
the measurements were done in the 1000-1800 cm-1 range for the homo-chiral LL-Trp2H+.
Quantum chemical calculations were performed in order to assign the experimental peaks
and interpret the recorded data. The conformational search was done with MD calculations
followed by structure optimization with DFT methods from the Gaussian 09 [132] and
Gaussian 16 [133] packages. Three types of intermolecular bonding [134] were tested: A:
NH•••N bond between the amino groups of the amino acids; B: NH•••O bond between the
protonated amino group and the oxygen of the carboxylic group of neutral Trp; Z: NH•••O
bond similar to type B but with Trp in zwitterionic form (Figure 3.14).
Figure 3.14: Types of bonding between protonated and neutral tryptophan tested in
calculations. Figure from paper V.
The population of the most stable conformers was calculated for room temperature (298.15
K) using equation 3.1:
𝑁𝑖
𝑁𝑡𝑜𝑡𝑎𝑙=
𝑒−𝐺𝑟𝑒𝑙
𝑅𝑇⁄
∑ 𝑒−𝐺𝑘
𝑅𝑇⁄𝑁𝑡𝑜𝑡𝑎𝑙
𝑘=1
, (3.1)
where Ni over Ntotal stands for the relative population of the conformer i, Grel and Gk are
relative free energies of the conformer i and k, respectively, R is the ideal gas constant, and
T is the temperature in Kelvin. The IR spectra predicted theoretically was scaled by a factor
36
of 0.955, which was obtained by fitting the theoretical spectra to the corresponding most
intense peaks in the experimental spectra.
Experimental and theoretical IRMPD spectra of homo-chiral LL-Trp2H+ are shown
in Figure 3.15.
Figure 3.15: Infrared spectrum of the homo-chiral LL-Trp2H+ for 1000-1800 cm-1 range
(left) and 2700-3750 cm-1 range (right) obtained (a, b) experimentally; computed with the
B3LYP/6-311++G**/GD3BJ method for (c, d) the most populated conformers; (e, f, g, h)
conformers with A type of interaction via NH•••N bond; (i, j) conformers with B type (via
NH•••O) and Z type (NH•••O bond with neutral Trp in zwitterionic form) of interaction.
Corresponding conformers are presented in Table 3.1. Figure from paper V.
Two intense peaks at 3575 cm-1 and 3520 cm-1 correspond to the OH stretch at the carboxylic
group and the NH stretch mode at the indole ring, respectively. The broad peak at
3212 cm-1 originates from the multiple vibrational modes in the amino groups. The peaks
at 3078 cm-1 and 2940 cm-1 correspond to the CH vibrations in the rings and backbones of
the amino acids, respectively (Figure 3.15). The mid-IR spectrum (Figure 3.15a) has
37
multiple overlapping bands. The peak at 1650 cm-1 at the C=O stretching region can be
distinguished. The populations, electronic and free energies of the most stable structures,
calculated with different methods, are shown in table 3.1.
Table 3.1: Populations, electronic and free energies of the most stable conformers for
homo- and hetero-chiral dimers of Trp. The data is shown for MP2/B3LYP, B3LYP and
M062X methods, calculated at 6-311++G** level of theory including GD3BJ empirical
dispersion.
Conformer
Method
MP2/B3LYP B3LYP M062X
Pop,
%
ER,
kcal/mol
GR,
kcal/mol
Pop,
%
ER,
kcal/mol
GR,
kcal/mol
Pop,
%
ER,
kcal/mol
GR,
kcal/mol
LL-A1 12.57
1.74 1.15 16.73 1.54 0.95 6.97
3.12 1.53
LL-A2 87.38 0 0 82.99 0 0 92.99 0 0
LL-CS1 0.02 5.16 5.11 0.08 4.14 4.09 0.03 5.49 4.84
LL-CS4 0.03 5.53 4.73 0.2 4.38 3.58 0.01 6.73 5.69
LL-B1 <0.01 18.48 14.51 <0.01 10.8 6.83 <0.01 14.05 11.78
LL-Z1 <0.01 20.84 17.34 <0.01 11.52 8.02 <0.01 16.09 12.11
LD-A1 88.44 0 0 45.09 0 0 59.65 0 0
LD-A2 11.56 2.56 1.21 54.91 1.23 -0.12 30.35 1.74 0.23
The conformational search showed that the conformers of Type A are the most
stable under our experimental conditions. The theoretical IR spectra of the conformers LL-
A1 and LL-A2 are in a good agreement with the experimental results (Figure 3.15). In
contrast, the population of the conformers LL-B1 and LL-Z1 is negligibly small (< 0.01 %).
The intense fingerprint peaks at 2820 cm-1 and 1390 cm-1 for the LL-B1 conformer and at
2875 cm-1 and 1375 cm-1 for the LL-Z1 conformer, respectively, were not detected in the
experiment (Figure 3.15). Therefore, these two types of conformers were not considered in
our further calculations. Our results suggest that the A type of the interaction is the most
stable one for the homo-chiral LL-Trp2H+. This finding is in a good agreement with
previously reported results [135].
The most populated homo-chiral conformers LL-A2 (about 80 % of the population)
and LL-A1 (about 10 % of the population) were compared with the structures found by Feng
et al. [135]. Both of the homo-chiral dimers found in our calculations (LL-A1 and LL-A2)
38
are bonded via two hydrogen bonds (Figure 3.16): the strong bond between the protonated
and neutral amino groups, and the weaker bond between the protonated amino group and
the nitrogen in the indole ring of neutral Trp. In contrast, besides the bonding between the
amino groups, the conformers LL-CS1 and LL-CS4 have the carboxyl group involved in the
intermolecular bonding (Figure 3.16), which result in the high intensity peak at 3250 cm-1
(Figure 3.15). This high intensity peak is not present in the experimental spectrum and
theoretical IR spectra for LL-A1 and LL-A2 conformers. Additionally, the population of the
LL-CS1 and LL-CS4 was found to be less than 0.04 %. Therefore, we concluded that these
conformers are not present under our experimental conditions.
Figure 3.16: The structures of most stable conformers for proton bound homo-chiral
dimers of Trp calculated with B3LYP/6-311++G**/GD3BJ method. The distances are
shown in Å. Figure from paper V.
Conformational search was also done for the hetero-chiral LD-Trp2H+ (Table 3.1).
The resulting most populated structures had the intermolecular interaction of A type
(Figure 3.17). The hetero-chiral conformers are bonded via two hydrogen bonds, one
between the amino groups, and the second one between the protonated amino group and
the nitrogen of the indole ring of the neutral Trp. The structures of A1 and A2 conformers
for homo- and hetero-chiral dimers are similar. The particular conformer-specific
39
difference can be pointed out for both diastereomers. The LL-A1 and LD-A1 conformations
have the indole rings in a close proximity to each other, whereas the conformers LL-A2 and
LD-A2 have remote arrangements of the rings (Figure 3.16 and 3.17).
Figure 3.17: The structures of the most stable conformers of the hetero-chiral dimers of
Trp calculated with the B3LYP/6-311++G**/GD3BJ method. The distances are shown in Å.
Figure from paper V.
The close proximity of the indole rings in the A1 type of conformation affects the indole NH
stretching mode of the neutral Trp. The free vibration of the NH mode is restricted, which
results in the red shift of this mode in the theoretical IR spectra (Figure 3.18).
40
Figure 3.18: IR spectra of homo-chiral LL-Trp2H+ and racemic mixture of Trp (a) obtained
in the experiment; (b) obtained in calculations with the B3LYP/6-311++G**/GD3BJ
method. Theoretical IR spectra computed with the B3LYP/6-311++G**/GD3BJ method for
two most stable conformers of (c) homo-chiral Trp2H+ dimer; (d) two most stable
conformers of hetero-chiral LD- Trp2H+ dimer. Figure from paper V.
In the conformers with the remote orientation of the indole rings (LL-A2 and LD-A2), the
NH stretch mode does not interfere with the other modes and consequently, the red-shift is
not present in the theoretical spectra.
The reproducible difference in the intensity of the NH stretch mode of the indole
ring was detected in the experiment, when the solution of LL-Trp2H+ and the racemic
mixture of Trp were used (Figure 3.18a). This difference might be a result of the higher
population of the A1 conformer for the hetero-chiral dimer (LD-A1 – 88 %) compared to
homo-chiral one (LL-A1 - 13 %). Interestingly, the intense peak at 3440 cm-1 present at the
theoretical spectra of LD-A1 was not observed in the experiment. This disagreement can be
explained if we consider that the red-shift predicted theoretically is not that pronounced in
41
the experiment and would result in the double peak of the NH mode of a higher intensity
for the A1 conformer.
Summarizing the results of this work, we found the structures of the most stable
conformers for homo- and hetero-chiral dimers of Trp. Both diastereomeric forms exist in
the A type of bonding via the amino groups of the amino acids. The conformers of the
diastereomers have similar structure, LL-A1 and LD-A1 with a close orientation of the
indole rings and LL-A2 and LD-A2 with the remote orientation of them. The red-shift of the
NH stretch mode was observed in theoretical IR spectra for the dimers with the indole rings
in close proximity to each other. This orientation restricts the free vibration of the NH mode
and results in the described effect. This might be an explanation for the difference in the
intensity of the NH mode, detected utilizing homo-chiral and racemic mixtures of Trp.
42
4. Conclusions and outlook
The focus of this work was the investigation of the fundamental interactions
responsible for chiral discrimination. The interactions of the protonated enantiopure amino
acids with chiral and achiral molecules in gas phase have been studied. In a contrast to
previously reported studies primarily using ion-trap-based techniques, here we used beam
type of experiments to investigate ion-molecule interactions.
High energy collisions of enantiopure amino acids with a chiral target restrict the
formation of the long-lived projectile-target complex. It leads to fragmentation,
independent of the chirality of the colliding pairs. The energy obtained in the 1 keV
collisions is sufficient to produce the fragmentation via multiple channels. The
fragmentation channels are in a good agreement with well-established fragmentation
models for the amino acids. Utilizing chiral and achiral target gases, the same
fragmentation in terms of the reaction channels and the relative abundance of fragments
were observed.
Low-energy collisions of protonated amino acids with chiral targets in the range of
meV - eV go via two processes: fragmentation and projectile-target adduct formation. The
energy dependence of the reactions was investigated. The fragmentation efficiency in a
single collision mode is significantly lower in comparison with high energy collisions. The
main reaction channel at the low energies is the formation of projectile-target adducts. Both
processes were found to be independent of the chirality of the reactants. The projectile-
target adduct formation in the single collisions mode without the pre-orientation of either
the projectile ion or the target molecule can lead to multiple different hydrogen bonded
complexes with similar structure and energy, which would limit the possibility to detect the
stereo-dependent effect.
The stereo-dependence was detected in collision-induced dissociation of the proton
bound diastereomeric adducts of Trp and 2-butanol formed in the electrospray prior to
collisions with the target gas. By varying the collision energy, the kinetics of the process was
studied. The hetero-chiral Trp-2-butanol is more stable than its homo-chiral counterpart
against CID. The DFT calculations have resulted in a structure that has more probable
interaction points for hetero-chiral adduct than the homo-chiral. The formation of the
adduct in the electrospray, prior to the collisions with the target, gives more possibility for
the specific intermolecular bonding to form, which can be different for each diastereomer.
However, at the same time, electrospray conditions can result in the raise of population for
the conformers close to the ground-state energy and the structures with the flexible
bonding. This was observed in our experiments with 1-phenylethanol. Replacing 2-butanol
with the bulkier 1-phenylethanol did not result in the enhancement of the stereochemical
43
effect, on the contrary, the effect disappeared. Three different amino acids have been tested
for diastereomeric adduct formation and the consequent CID reactions, yet no chiral
recognition was observed. The structures of diastereomeric adducts obtained with the DFT
calculations have the hydrogen bond between the protonated amino group of amino acid
and the hydroxyl group of alcohol. This single intermolecular bonding permits the flexibility
in relative orientation of the constituents, and is not restricted to a rigid confirmation with
multiple points of interaction. The structures of hetero-chiral adducts were found to be
more stable in terms of free energy. Yet, the additional intermolecular interactions were not
observed for any pair of amino acid and 1-phenylethanol and might be the main reason of
the null result.
The proton bound diastereomers of Trp were studied with IRMPD spectroscopy and
DFT calculations. The structure of the most stable conformers has been revealed. The
bonding between the amino groups of protonated and neutral amino acids was found to be
the main type of intermolecular interaction in protonated Trp dimers under the presented
experimental conditions. The two most stable conformers with two intermolecular
hydrogen bonds were found for homo- and hetero-chiral dimers. The fingerprint peaks were
found for the conformers in the calculated IR spectra. The higher population of the
particular conformer for the hetero-chiral dimer might be an explanation for the different
IRMPD spectra obtained in the experiment. Additional more precise calculations with
anharmonic frequencies at different initial temperatures would be desirable for better
understanding the experimental results. However, it is computationally very demanding.
The importance of the conformational analysis and conformation-specific types of
the experiment is an important requirement to be able to unambiguously interpret the
experimental results and compare them with the theoretical calculations. The possibility to
cool-down the chiral ions in the collision and spectroscopy experiments would be beneficial
for the study of stereo-dependent interactions. Furthermore, the experiments at cryogenic
temperatures would bring an important information about the interactions of chiral
molecules in the universe and might shed a light on the fundamental question of the origin
of homochirality in life.
44
Acknowledgements I would like to say thank you to everyone whom I have met during my PhD time. All
of you have made this time very special. I am grateful to my supervisor Mats Larsson. Mats,
thank you so much for the opportunity to work with you, for your trust in me, and your
endless support and understanding. I have learnt so much from you, not only in science but
also in life. My co-supervisor Richard, thank you for your great input in my research and in
my teaching experience. It was lots of learning and fun. My second co-supervisor Vitali, I
really appreciate your supervision and very interesting and fruitful experiments-
discussions. I will always remember how we operated the free electron laser, equipped with
several books of manuals, and our French lunches in “Chromophore”. My mentor Åsa,
thank you for your advice and your support. It was very helpful to know that I always can
talk to you and get very useful suggestions about my PhD. Before I started my PhD studies
I got the priceless and unique experience in Roman Zubarev lab at Karolinska Institute.
Roman, thank you for giving me this chance, for your care, support and guidance. Kostya
Chingin, I will always remember our discussions next to Orbitrap with plasma gun attached
to it. Your experience and your supporting words always help me.
I would like to express my gratitude to our collaborators and people who was actively
involved in work. Kostiantyn Kulyk, Einar Uggerud, Mauritz Ryding, Henning Zettergren,
Henning T. Schmidt, Henrik Cederquist, Michael Odelius, Mark H. Stockett, John D.
Alexander, Mathias Poline, Göran Sundström, Philippe Maitre, Estelle Loire, Vasyl Yatsyna,
Stefano Stranges and his group.
Colleagues and friends from Physics department Jesper, Ting, Ida, Emili, Kess,
Sifiso, Fredrik, Axel, Tej, Nader, Oliver, Sergey, Gustav, Yurij, Anna, Nathalie, Daniel,
Emma, Linda, Moa, Shree, Mikael Wolf, Tor, Markus, Semeli, Mikica. Thank you all. You
made my PhD time very special.
I greatly appreciate support and inspiration from my friends and very important
people: Oleksandr, Victoria, Sifiso, Alexey, AlexDL, Evgenii, Mirko, Alösha, Joachim and
Simone, Conran, Armando and Cindy, Nandi, Franchesca, Laura and Gustav, Families
Sidora, Gorianij, Neshta, Pivovarov, Dun’ and Goryashko.
My brother Oleg. I am very happy that we had that one month together during my
studies and rediscovered each other. Thank you for trust in me, your visits, support and
help. I would like to say many thanks to my mother Olga and my father Vladimir. Мои
дорогие родители, без вашего безграничного доверия, поддержки, заботы и любви
это было бы невозможно. Спасибо за ваше открытое сердце и прогрессивный,
молодой подход ко всему новому, готовность быть рядом со мной и нами, когда это
так необходимо. Спасибо моим бабушкам и дедушкам: Эльвире, Фаине, Анатолию и
45
Юрию. Ваш пример, невероятная жизненная энергия и мудрость всегда меня
поддерживают, вдохновляют и помогают. Мои дорогие сестрички Оля и Ира, я вас
очень люблю. Спасибо, что вы всегда со мной. During my PhD time, my family has
expanded and thereby I have had an incredible opportunity to experience Austria, Austrian
people, their traditions and life. Lieber Gerald, liebe Nathalie, Anita und Hans, Kati, Günter,
die ganze Familie Steiner und Fischereder! Es ist mir eine große Freude und Ehre euch
kennen zu lernen und Mitglied der Familie zu sein. Danke für die Freundlichkeit,
Gastfreundschaft und Unterstützung. I would like to express my gratitude to every member
of the Ukrainian-Russian-Austrian family. Thank you for your support, care and influence.
You are the best.
My wife Eva Maria, my Darling and my Love. Life with you is a Dream. Thank you
for non-stop happiness that I feel since I have met you. For a new world that you open to
me every day. For your endless support, kindness, patience and Love. My little princesses
my daughters Annika and Violetta. You are my two stars. You teach me every day something
new. And you show me that we are all kids, that just do more and more complicated things
every day.
46
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