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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