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8-2016
Mass spectrometric characterization of remotelycharged amino acids and peptidesDamodar KoiralaPurdue University
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PURDUE UNIVERSITY GRADUATE SCHOOL
Thesis/Dissertation Acceptance
To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
Damodar Koirala
MASS SPECTROMETRIC CHARACTERIZATION OF REMOTELY CHARGED AMINOACIDS AND PEPTIDES
Doctor of Philosophy
Paul Wenthold
Hilkka I. Kenttämaa
Yu Xia
Paul Wenthold
Timothy Zwier
Timothy Zwier 04/15/2016
MASS SPECTROMETRIC CHARACTERIZATION OF REMOTELY CHARGED
AMINO ACIDS AND PEPTIDES
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Damodar Koirala
In Partial Fulfillment of the
Requirements of the Degree
of
Doctor of Philosophy
August 2016
Purdue University
West Lafayette, Indiana
ii
To Koirala Family
iii
ACKNOWLEDGMENTS
It certainly feels great to have the opportunity to thank individual or group that
inspired, helped, supported and cared me to be where I am today. I am thankful to God
for sending all these amazing people to be part of my life.
I want to begin thanking my parents for the quality education and their supported.
Being the youngest in the family, my brother and sister always made sure I am not losing
track on my passion. I am also thankful to my relatives who have helped our parents
when I am away from them.
I would like to thank my friends and advisors at University of Wisconsin-
Superior. The parties and walk to the bars with friends are always hinging on my mind.
The food and gathering at Tom Markee’s house was always great. I am very thankful for
Tom for being my mentor, you are a great teacher. I still can’t believe your wife Sue is
not with us anymore, she has been truly missed. I would also like to thank Profs Waxman
and Lane for their inspiration on learning chemistry in class and in laboratory.
I feel blessed to have a wife who is with me on every twist and turns. She has
helped me throughout my Ph.D. career starting from OP. We are blessed to have a
wonderful daughter who is always there to refresh us and make us feel like we are in
different world.
iv
The Nepali community at Purdue is a very helpful community. It consists of very
helpful and educated people. They make me feel like I have never been away from my
family. They are always there during the time of difficulties. We had lots of fun together,
lots of parties, sports, travelling, casinos and celebrations. I had pleasure serving as a
treasurer (2014-15) and a president (2015-16) of Nepali Society at Purdue.
I am thankful for the colleagues at Department Chemistry at Purdue. Class of
2011 was great, we might have more parties and fun than any other group we had our
own club ‘Club 1106’. Bharat and Manish might be ones who were closer to me during
my stay at Purdue. Bharat and I shared lots of moment together from good to bad. We
prepared together for cumes, analytical seminar and OP. Manish and I also shared lots of
moment together. I spend most of first year at the apartment that he and Janak shared.
And we all moved to next apartment and lived together during our second year at Purdue.
Those moments were best, good food, FIFA games and NBA double header on TNT. I
also want to thank Manish and Neha for helping me on dipeptide synthesis. In addition, I
want to thank Hari Khatri dai for his support and help in several chemical synthesis and
NMR analysis. Hari dai devoted his valuable time on analyzing my sample and also
setting up some synthesis in this lab hood. Thank you all.
I am grateful for the support I received from my major advisor, Prof Paul G
Wenthold, during my stay at Purdue University. I must say he is a good man with big
brain and bigger heart. He has great passion in Chemistry and science in general. I can
never forget the ideas, motivations and enthusiasm that Paul brought to one on one or
group meetings. I always had positive productivity after meeting with him. Thank you.
v
I would like to thank the former Wenthold Lab members that I worked with. Dr.
Nathan J. Rau was my mentor who taught me how to use flowing afterglow. I was very
inspired by your knowledge on Chemistry and your dedication to work perfectly.
Besides, the time we spend together at ASMS conference in Vancouver. Do you
remember uphill from conference hall to the hotel, it was very steep, right? I did not
worked with Dr. Ekram Hossain but we spend lots of time together in lab and outside.
Ekram is a very intellectual person with lots of courage to try new stuff. I am sure you
will be big time professor one day. One moment I recall being with you is when Bharat
dropped you to the wrong airport in Indianapolis. Beside these two, I enjoyed working
with undergraduate Deon Turner. Actually, he synthesized the amino acid studied in
chapter 6 of this thesis, and did some mass spectrometry studies on that compound. I
wish him good luck for bright future.
Now, I would like to remember current Wenthold Lab members. I was very happy
when Chris Haskin joined our lab because I was by myself for long period of time. Chris
changed the structure of lab by cleaning and organizing old stuff. He is junior to me but I
am surprised how smart he is, even Paul would ask him on organic reactions, Wow. Babu
joined our lab within a month after Chris joined, so then we became triplet. My friends
teased us using MS terminologies “single-quadrupole changed to triple-quadrupole”.
Babu has very enthusiastic personality with diverse knowledge on the field of Chemistry.
He looks like a youngster with lots of energy but she always tells me that he is older than
me, which I have hard time believing. If I was not graduating soon then I am sure we
would have collaborated and have at least a co-authored peer reviewed article. I also
enjoyed being around and working together with undergraduate students; James
vi
Langford, Bryan Smith and Chris Brown. They are all smart guys with lots and lots of
talent on them. They all know world history almost in detail. James is very good at
communicating and telling stories about his research. He is a person who I witnessed
struggled a lot at the beginning of his research career and improved day by day to be a
systematic researcher. I worked with Bryan only for a semester when we struggle a lot on
synthesis. When Chris Haskin joined the group, Bryan is having lots of success and has
become independent researcher as well. The meeting at the far was awesome. I helped
Chris Brown on several incidences but never worked together in a project. In my
understanding, Chris Brown had good knowledge on how researches are performed even
before he joined or started using our lab. So when free, we spend most of our time on
discussion and future goal. Overall, I enjoyed the company of current lab member.
Thank you all for your continuous support and believing on me. I will do my best
to keep all the promises.
vii
TABLE OF CONTENTS
Page
LIST OF TABLES ...............................................................................................................x
LIST OF FIGURES ........................................................................................................... xi
ABSTRACT ..................................................................................................................... xiv
CHAPTER ONE: GUIDE TO DISSERTATION ................................................................1
1.1 Guide to Dissertation ...............................................................................................1 1.2 References ................................................................................................................3
CHAPTER TWO: REVIEW ON GAS-PHASE STRUCTURES OF AMINO ACIDS .....4
2.1 Introduction ..............................................................................................................4 2.2 Experimental Techniques.........................................................................................8 2.3 Computational Studies ...........................................................................................10 2.4 Conformations of AAs in Gas Phase .....................................................................11
2.4.1 Group 1: AA with R = H i.e. (Gly) ............................................................. 12 2.4.2 Group 2: AAs with Aliphatic Side Chains ....................................................16 2.4.3 Group 3: AAs with Oxygen or Sulfur on Side Chain ...................................21 2.4.4 Group 4: AAs with Amine Substituted Side Chain ..................................... 24 2.4.5 Group 5: AAs with Carbonyl Substituted Side Chain ................................. 25 2.4.6 Group 6: AAs with Aromatic Side Chain .................................................... 26
2.5 Stabilization of Zwitterionic AAs in Gas Phase ....................................................28 2.6 Conclusion .............................................................................................................31 2.7 References ..............................................................................................................32
CHAPTER THREE: EXPERIMENTAL APPROACHES ................................................37
3.1 Introduction ............................................................................................................37 3.2 The Flowing-Afterglow Triple-Quadrupole Mass Spectrometer ..........................38
3.2.1 The Ion Source ..............................................................................................38 3.2.2 The Mass Analyzer .......................................................................................40 3.2.3 Ion Detection Region ....................................................................................41
viii
Page 3.3 The LCQ-Deca Mass Spectrometer .......................................................................42
3.3.1 The Ion Source ..............................................................................................42 3.3.2 The Mass Analyzer .......................................................................................43 3.3.3 Ion Detection Region ....................................................................................46
3.4 References ..............................................................................................................47
CHAPTER FOUR: THE FLOWING AFTERGLOW STUDY OF PYRIDINE N-OXIDE NITRENE RADICAL ANIONS ......................................................................48
4.1 Introduction ............................................................................................................48 4.2 Computational Detail .............................................................................................53 4.3 Results ....................................................................................................................53
4.3.1 Reactivity Studies .........................................................................................54 4.3.1.1 Reaction with NO .............................................................................56 4.3.1.2 Reaction with CS2 .............................................................................58 4.3.1.3 Comparison of Isomers: Oxygen Nucleophilicity ............................64
4.4 Conclusion .............................................................................................................67 4.5 References ..............................................................................................................68
CHAPTER FIVE: MASS SPECTROMETRIC STUDY OF THE DECOMPOSITION PATHWAYS OF CANONICAL AMINO ACIDS AND Α-LACTONES IN THE GAS PHASE ...............................................................................................................................70
5.1 Introduction ............................................................................................................70 5.2 Experimental ..........................................................................................................74 5.3 Synthesis ................................................................................................................75 5.3.1 (Z)-4-(3-ethoxy-3-oxoprop-1-en-1-yl)benzenesulfonate(deprotonated
ethyl 4-sulfo-cis-cinnamate) .................................................................................75 5.3.2 4-(acryloyloxy)benzenesulfonate (PASO3
) .................................................76 5.4 Computational Methods .........................................................................................76 5.5 Results ....................................................................................................................77 5.5.1 Dissociation of PheSO3
...............................................................................79 5.5.2 Dissociation of the α –Lactone, αLSO3
......................................................86 5.6 Discussion ..............................................................................................................88 5.6.1 Amino Acid Dissociation ..............................................................................90 5.6.2 Dissociation of the α –Lactone .....................................................................94 5.7 Conclusions ............................................................................................................99 5.8 References ............................................................................................................101
ix
Page
CHAPTER SIX: EFFECT OF CHARGE POLARITY ON AMINO ACID DISSOCIATION..............................................................................................................107
6.1 Introduction ..........................................................................................................107 6.2 Synthesis ..............................................................................................................108 6.3 Results and Discussion ........................................................................................108
6.3.1 Dissociation of PheNMe3+ .........................................................................109
6.3.1.1 Neutral Loss of Mass 17 Da and Formation of m/z 206 ion ...........111 6.3.1.2 Neutral Loss of Mass 44 Da and Formation of m/z 179 ion ...........113 6.3.1.3 Neutral Loss of Mass 45 Da and Formation of m/z 178 ion ...........118 6.3.1.4 Neutral Loss of Mass 74 Da and Formation of m/z 149 ion ...........118 6.3.1.5 Neutral Loss of Mass 87 Da and Formation of m/z 136 ion ...........118
6.4 Conclusion ...........................................................................................................119 6.5 References ............................................................................................................124
CHAPTER SEVEN: MOBILE C-H PROTONS IN A PROTON DEFICIENT PEPTIDE .......................................................................................................................126
7.1 Introduction ..........................................................................................................126 7.2 Experimental ........................................................................................................130 7.3 Synthesis ..............................................................................................................131 7.3.1 Synthesis of Amino Acid Esters .................................................................131 7.3.2 Synthesis of Gly-Phe*OH and Gly-Phe*OMe Dipeptides .......................131 7.3.3 Synthesis of Phe*-GlyOH and Phe*-GlyOMe Dipeptides .......................132 7.3.4 Synthesis of Diketopiperazine ....................................................................132 7.3.5 Synthesis of Gly-Phe* Oxazolone ..............................................................133 7.4 Results and Discussion ........................................................................................134
7.4.1 Analysis of Gly-Phe*OH and Phe*-GlyOH .............................................134 7.4.2 Analysis of Gly-Phe*OMe and Phe*-GlyOMe ........................................136 7.4.3 Deuterium Labelled Phe*-GlyOMe ...........................................................142 7.4.4 Computational Results ................................................................................146
7.5 Conclusion ...........................................................................................................148 7.6 References ............................................................................................................149
VITA ................................................................................................................................152
PUBLICATIONS .............................................................................................................153
x
LIST OF TABLES
Table Page
2.1Significance of 20 α-amino acids .............................................................................. 6
4.1Reaction Efficiencies and Product Branching Ratios for Reactions of 3PNO and 4PNO with NO and CS2 .................................................................... 56
5.1Computed Reaction Energies for Dissociation Pathways of PheSO3 and
Phe .......................................................................................................................... 89
7.1Calculated Relative Energies of b2 Ions ................................................................ 147
xi
LIST OF FIGURES
Figure Page
2.1 Structure of naturally occurring 20 α-amino acids ................................................... 5
2.2 The 4 types of possible intramolecular hydrogen bond in AAs ............................. 11
3.1 The flowing-afterglow triple-quadrupole mass spectrometer ................................ 38
4.1 Resonance effect due to N-Oxide motif (Left) and the resonance structures of the π2α and α2π states of 4PNO (Right) ............................................................ 52
4.2 The m/z 92 region of the mass spectra of 4PNO before (dashed) and after (solid) addition of CS2 ............................................................................................ 60
4.3 The m/z 92 region of the mass spectra of 4PNO reaction with CS2 (dashed) and after addition of NO (solid) ............................................................... 61
4.4 Calculated Mullikin charge distributions in 3PNO , 4PNO and pyridine-n-oxide. Charges on hydrogen have been summed into the heavy atoms. ............ 66
5.1 a) ESI and b) CID mass spectra of PheSO3. The CID for spectrum was carried out with a normalized collision energy of 25%. ......................................... 78
5.2 Low-energy conformations of the amino acid moiety of PheSO3 and their relative energies, in kcal/mol, computed at the B3LYP/6-31+G* and MP2/6-311+G** (in parentheses) level of theory .................................................. 79
5.3 CID spectra of m/z 227 ions obtained a) by CID of PheSO3 , and from sulfonated b) cis and c) trans cinnamic acid ........................................................... 83
5.4 Calculated energetics for the dissociation of PheSO3 (X = SO3) and Phe
(X = H). Values correspond to electronic energies and geometries calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of theory a) not a stable geometry; corresponds to an amino acid geometry the same as that for zwitterionic PheSO3; b) computed utilizing a 3 kcal/mol barrier for the reverse reaction, obtained from a relaxed surface scan; see Supporting Information. ............................................................................................................ 91
xii
Figure Page
5.5 Calculated energetics for the dissociation of αLSO3 (X = SO3
) and the non-sulfonated derivative (X = H). Values correspond to electronic energies and geometries calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of theory, unless indicated a) not a stable geometry; corresponds to the geometry of the lactone with the CO2 removed ....................... 96
6.1 The 7 low energy conformations of PheNMe3+. Relative energies are
shown in kcal/mol ................................................................................................ 109
6.2 CID spectra of a) PheNMe3+ with m/z 223, b) d3-PheNMe3
+ with m/z 226 ....... 110
6.3 CID spectra of m/z 206 obtained from a) CID of PheNMe3+, b) authentic
cinnamic acid (1) .................................................................................................. 112
6.4 CID spectra of a) m/z 179 obtained from CID of PheNMe3+, b) 2 with m/z
179, c) d2-2 with m/z 181 ..................................................................................... 114
6.5 CID spectra of m/z 164 obtained from a) 2, b) PheNMe3+ .................................. 115
6.6 PES for decarboxylation of PheNMe3+, PheSO3
and Phe. The barriers and the molecular energy were were calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of theory. ........................................................ 117
6.7 Proposed Fragmentation Pathways of PheNMe3+................................................ 120
6.8 Possible rearrangement products of PheNMe3+ and their relative energies
in kcal/mol calculated at the B3LYP/6-31+G* level of theory .......................... 121
6.9 CID spectrum of Ia with m/z 223 ........................................................................ 123
7.1 Formation of diketopiperazine from dipeptide with cis amide bond (top) and formation of oxazolone from dipeptide with trans amide bond (bottom) ..... 128
7.2 Mass Spectra: a) CID of Gly-Phe*OH and b) CID of Phe-Gly*OH ................. 135
7.3Proposed mechanism for formation of common structure from Phe*-GlyOH and Gly-Phe*OH .......................................................................... 136
7.4 Mass Spectra: a) CID of Gly-Phe*OMe and b) CID of Phe*-GlyOMe ............. 138
7.5 Mass Spectra: CID of b2 ions a) MS3 spectra of Gly-Phe*OMe and b) MS3 spectra of Phe*-GlyOMe c) CID of 1 d) CID of 2 ................................. 140
7.6 Mass Spectrum: CID of Phe*-Gly (1-13C) OMe .................................................. 142
xiii
Figure Page
7.7 Mass Spectra: CID of deuterated sample a) d3-Phe*-GlyOMe b) Phe*-Gly (2, 2-d2) OMe c) d5- Phe*-Gly (2, 2-d2) OMe ............................ 145
7.8 Proposed fragmentation pathway of Phe*-GlyOR’ ............................................ 146
7.9 Lowest energy structures of 1, 3, 4, 2, and 5 respectively ................................... 148
xiv
ABSTRACT
Koirala, Damodar. Ph. D., Purdue University, August 2016. Mass Spectrometric Characterization of Remotely Charged Amino Acids and Peptides. Major Professor: Paul G. Wenthold. Ion–molecule reactions in a flowing afterglow are used to examine the
electronic structure of 3- and 4-pyridinylnitrene-n-oxide radical anions. Reactions with
nitric oxide are generally similar to those reported previously for other aromatic
nitrene radical anions. In particular, phenoxide formation by nitrogen–oxygen
exchange is observed with both isomers. Oxygen atom abstraction by NO is also
observed with both isomers. Very significant differences in the reactivity are observed
in the reactions of the two isomers with carbon disulfide. The reactivity of the 3-n-
oxide isomer with CS2 is similar to that observed previously for nitrene radical anions,
and reactions of the n-oxide moiety are not observed, similar to what is expected based
on solution chemistry. The 4-n-oxide isomer, however, undergoes many reactions,
including oxygen atom and oxygen ion transfer and sulfur–oxygen exchange, that
involve the n-oxide oxygen. The increased reactivity of the oxygen is attributed to
increased charge density at the oxygen due to pi electron donation of the nitrene anion
in the para position.
The dissociation pathways of gas-phase amino acids with a canonical (non-
zwitterionic) α-amino acid moiety are studied by using mass spectrometry.
xv
Investigation of the canonical amino acid moiety is possible because the ionized
amino acid has a charge center that is separated from the amino acid, and dissociation
occurs by charge-remote fragmentation. The negatively charged amino acid is found
to dissociate only by loss of NH3 upon collision-induced dissociation to form a
substituted α-lactone. The collision-induced dissociation spectrum of the positively
charged amino acid is complex with possibly 5 different fragmentation pathways. The
results on negatively charged amino acid, para-sulfonated phenylalanine (Phe*), show
that remote ionic groups can be used as mostly inert charge carriers to enable mass
spectrometry to be used to investigate the gas-phase physical and chemical properties
of different types of functional groups, including amino acids.
The b2 ion formed upon charge remote fragmentation of dipeptides, Phe*-
GlyOH and Gly-Phe*OH, is characterized using LCQ-Deca mass spectrometry.
Comparison with authentic samples confirmed the lack of diketopiperazine or
oxazolone on b2 ion and electronic structure calculation suggested that the b2 ion is
oxazolone-enol.
1
CHAPTER ONE: GUIDE TO DISSERTATION
1.1 Guide to Dissertation
Mass Spectrometer (MS) is a quantitative tool used to measure the mass-to-charge
ratio of ions. For more than a decades, our group has used a home-built flowing-
afterglow triple-quadrupole MS1,2 to characterize the electronic structure of nitrene
anions.3 Nitrenes are a fascinating because although isoelectronic to carbenes, they show
very different reactivity.4-6 Recently, our group has started to use a commercial LCQ-
Deca MS7 to investigate the charge remote fragmentation8,9 of amino acids and dipeptides.
10
This thesis describes the gas-phase characterization of pyridine n-oxide nitrene,
and charge-remote fragmentation of amino acids and dipeptides using mass spectrometers
(MS) as an analytical tool. Chapter 2 features a literature review on the gas-phase
structure of the α-amino acids. It addresses the interactions present on low energy
conformation of gaseous amino acids and how the different side chains on amino acids
affect those interactions.
All the experimental results in this thesis were obtained from a home-built
flowing-afterglow triple-quadrupole MS,2 or a commercial LCQ-Deca MS.7 Chapter 3
has the detail explanation on different components of these instruments.
2
In Chapter 4, we report the investigation on reactivity of 3- and 4-
pyridinylnitrene-n-oxide radical anions with O2, CO2, NO and CS2. We discovered that
these isomer react differently only with CS2 where N-oxide of 4-PNO. participates to
yield CS2O- product. The dissimilarity in reactivity is attributed to the resonance structure
of 4-PNO. where charge is in N-Oxide, which is not possible in 3-PNO. isomer.
Chapters 5 and 6 describe the positive and negative charge-remote fragmentation
of phenylalanine, PheSO3 and PheNMe3+, respectively. Techniques like isotope
labelling, comparison with authentic sample and electronic structure calculations are
implemented to characterize fragmentation ions and neutrals. We discovered PheSO3
has a single fragmentation pathway whereas PheNMe3+ has multiple fragmentation
pathways. The discrepancy in dissociation of PheSO3 and PheNMe3+ has been attributed
to the possibility on rearrangements of PheNMe3+ prior to dissociation.
Chapter 7 focuses on the characterization of the b2 ions formed upon dissociation
of dipeptide containing para-sulfonated phenylalanine and glycine. We discovered these
dipeptides rearrange to common structure before fragmentation. Comparison with
authentic samples confirmed the lack of diketopiperazine or oxazolone on b2 ion and
electronic structure calculation suggested that the b2 ion is oxazolone-enol.
3
1.2 References
(1) Marinelli, P.; Paulino, J.; Sunderlin, L.; Wenthold, P.; Poutsma, J.; Squires, R. Int. J. Mass Spectrom. Ion Processes 1994, 130, 89.
(2) Graul, S.; Squires, R. Mass Spectrom. Rev. 1988, 7, 263.
(3) Rau, N.; Welles, E.; Wenthold, P. J. Am. Chem. Soc. 2013, 135, 683.
(4) Platz, M. Acc. Chem. Res. 1995, 28, 487.
(5) Horner, L.; Christma.A . Angew. Chem. Int. Ed. 1963, 75, 707.
(6) Belloli, R. J. Chem. Educ. 1971, 48, 422.
(7) Thermo Electron Corporation: San Jose, CA.
(8) Cheng, C.; Gross, M. Mass Spectrom. Rev. 2000, 19, 398.
(9) Adams, J. Mass Spectrom. Rev. 1990, 9, 141.
(10) Koirala, D.; Kodithuwakkuge, S.; Wenthold, P. J. Phys. Org. Chem. 2015, 28, 635.
4
CHAPTER TWO: REVIEW ON GAS-PHASE STRUCTURES OF AMINO ACIDS
2.1 Introduction
Amino acids constitute a class of organic molecules containing carboxylic acid (-
COOH) and amine termini (-NH2). One way to classify amino acids is according to the
location of core structural functional groups such as alpha- (α-), beta- (β-), gamma- (γ-)
or delta- (δ-) amino acids. The α-amino acids are of particular interest because they are
proteinogenic, meaning they constitute the building blocks of peptides and proteins.
Although amino acids are abundantly found in nature, proteins are constructed from a set
of 20 naturally occurring α-amino acids, Figure 2.1. The generic formula for an α-amino
acid is RCH(NH2)COOH where the amine group (-NH2) and variable side chain ( R ) are
attached to the α-carbon of the carboxylic acid group (-COOH). Proteinogenic amino
acids are indispensable agents of biological function since some proteins functions as
enzymes, some as antibodies, and others provide structural supports. In addition, these
amino acids may have non-protein functions as neuro-transmitters or as precursors of
important neuro-transmitters or hormones, Table 1.1. For these reasons, the intrinsic
properties of the 20 naturally occurring α-amino acids have attracted the interest of a
number of researchers over the past several decades. For simplicity, α-amino acid will
here with be referred to as AA.
5
Figure 2.1. Structure of naturally occurring 20 α-amino acids
6
Table 2.1: Significance of 20 α-amino acids
AA Significance
Gly Essential for the synthesis of nucleic acids, bile acids, and porphyrins.
Ala Involves in the metabolism of the vitamin pyridoxine.
Val Incorporate into poteins and enzymes to help dictate the tertiary structure of the macromolecules. Leu
Ile
Pro Acts as a major component of the protein collagen and the connective tissue.
Ser Play an important role in porphyrins metabolism. Thr
Cys Metabolisms of coenzyme A, heparin, biotin and lipoic acid.
Met Initiates translation of messenger RNA.
Lys Used at the active sites of enzymes. Arg
Asn
Present outside proteins and enzymes interacting with the surroundings. Asp Glu Gln
His Responsible for the biosynthesis of neurotransmitter namely histamine.
Phe Play key roles in the biosynthesis of neurotransmitters. Trp
Tyr Synthesis of thyroid hormones and melanin biological pigments.
The function of proteins and their multidimensional structures are highly
dependent upon the conformation of their constituent AAs. Therefore, the knowledge of
the shape and the conformation of AAs is of great interest in a variety of fields, ranging
from biology and chemistry to pharmacology and molecular modeling. AAs have been
7
studied extensively in the condensed phase 1-4 and in the solution 5-7 but less so in the gas
phase.8,9 AAs exist in equilibrium between a doubly-charged species of zwitterions (Z),
RCH(NH3+)COO-, and neutral canonical forms (N), RCH(NH2)COOH, Scheme 2.1.8,10-
13 They are predominately present as zwitterions in condensed phase due to the strong
electrostatic, intermolecular, and polarizing interactions with the environment. However,
the lack of these intermolecular interactions favors canonical forms of AAs in gas phase
8,9,13. Since studies in condensed phases are affected by intermolecular interactions,
conformational preferences can be biased by the environment.14 The advantage of gas-
phase studies is that the gas-phase data can be contrasted with the theoretical models and
the molecules can be studied in detail with an optimized interplay between experiment
and theory.15
Gas phase study of isolated AAs creates an opportunity to provide information on
the neutral canonical forms of AAs present in peptide side chains. In addition, the
inherent molecular properties are provided, free of the intermolecular interactions
occurring in the condensed phase where AAs are bipolar zwitterionic species. Although
the intrinsic properties of isolated AA are of great interest, understanding the role of
solvent is also equally important since biological molecules including proteins and AAs
Scheme 2.1
8
exist in solution in living organisms.16 Hence, AAs are studied in gas-phase either in
isolated form or with a controlled number of solvent molecules. In particular, because
AA-AA intermolecular interaction can be neglected, the effect of solvation on the
configuration of an AA can be compared between experiment and theory. Most of the
experimental and theoretical studies of these types are designed to determine AA
configuration in the presence of a known number of solvent molecules.8,17-21 The latest
review by Bouchoux10 covers the results regarding neutral AA configuration and basicity
in the gas phase. This review summarizes the experimental and theoretical findings on
low energy conformers of canonical AAs, as well as addressing the chemical
environment required for stability of zwitterionic structures.
2.2 Experimental Techniques
Several techniques have been implemented to study the conformations of
canonical AAs is gas phase. Samples are first sublimed into gas phase and then analyzed
using spectroscopic methods.
Conventionally, solid sample on a uniformly heated stainless steel surface parallel
to a high vacuum chamber where desired analysis can be performed. This heating method
has presented considerable difficulties on creating gaseous AAs due to the high melting
points and associated low vapor pressures of AAs. Nonetheless, conventional heating
method has been employed for the analysis of gaseous Gly11,22-24, Ala11,22,Val 11,25, Cys22,
Thr22, Met22, Pro11,25, Phe25, Ile25 and Leu25.
An alternative to conventional heating is laser ablation. In this method, the fast
desorption produced by the energy of a laser pulse prevents thermal decomposition
9
normally caused by conventional heating. An Nd-YAG laser is used to ablate solid
samples, which are then seeded in the carrier gas and are expanded supersonically in an
analysis chamber.
Until the introduction of laser-ablation techniques, a gas-phase spectrum was only
available for solids with sufficient vapor pressure and thermal stability. Hence, there was
a lack of information on the structures of most AAs and microsolvation clusters due to
the vaporization difficulties associated with the aforementioned high melting points and
thermal instability. Several AAs like Gly, Ala26, Val16, Ile27, Leu28, Ser14, Cys29, Phe30,
Pro31,32, Thr14, Asp33 ,Asn30, Glu34, His35,Trp36, have now been studied in gas phase by
this means.
Several spectroscopic methods have been implemented to study gaseous
aminoacids. Microwave spectroscopy has been implemented Gly, Ala22,26, Val16, Ile27,
Leu28, Ser14, Cys29, Phe30, Pro31,32, Thr14, Asp33, Asn30, Glu34, His35 and Trp36 in gas
phase. Infrared spectroscopy has been used to study AAs like Gly22, Ala22, Thr22, Cys22,
Met22, Val25, Pro25, Phe25, Ile25 and Leu25. Raman spectroscopy 37,38 and electron
diffraction spectroscopy 39 also are implemented to study amino acids in gas phase.
10
2.3 Computational Studies
Each experimental study is conducted along with a complementary
computational study. Computational studies are used to find the low energy conformers
of the AA and the desired parameter associated with them. For example, in case of
microwave spectroscopy, the rotational constants for several low energy conformers are
predicted computationally and compared with experimental results. Computational
studies assist on qualitative and quantitative analysis of conformations present in a
gaseous sample of the AA. 23,38,40,41
Although computational studies help to interpret the experimental findings, it can
easily mislead to a false conclusion. For instance, the results for gas-phase study of
canonical Met22,42 were interpreted based on the conformations that were later discovered
to be more than 10 kJ/mol above the most stable conformation. So the conclusions drawn
at that time are now invalid10.
On the other hand, a false conclusion could be drawn due to false or insufficient
experimental outcomes. For instance, the rotational constant obtained from the
microwave spectroscopy of Gly matched with the second most stable conformer of Gly
23,43 suggesting this is the stable conformation of Gly is gas phase. However, it was later
discovered that it was not possible to ascertain which conformer was the main one from
that investigation because the intensities of the microwave spectra strongly depend on the
magnitude of the dipole moment of the molecule and not on the abundance of the
conformer in the gaseous sample 12,44. But with some modifications to the instrument, a
convincing result between experimental and theoretical studies of gaseous Gly
11
conformers was drawn.45 Therefore, systematic experimental and theoretical studies are
essential to make a conclusive decision on preferred conformation of gaseous AAs.
2.4 Conformations of AAs in Gas Phase
AAs are very flexible molecules, and their conformational landscape is
characterized by a delicate balance of covalent and non-covalent interactions, which may
result in a large number of low-energy conformers.26 The most important interaction
present in gaseous AA is intramolecular hydrogen bonding. There are four common
intramolecular hydrogen bonding present in AA, Figure 2.2.10,16 They are: 1) between the
acidic hydrogen and the carbonyl oxygen, 2) between acidic hydrogen and neighboring
basic site, 3) between N-H hydrogen and carbonyl oxygen, and 4) N-H hydrogen and
hydroxy oxygen as shown in Figure 2.2. The combinations of these interactions create
three unique interactions namely types I, II and III as shown in Figure 2.2. These
notations are used throughout this review for classification of interaction present in AA.
The following section describes the structure of AAs based on the side chain.
Figure 2.2. The 4 kinds of possible intramolecular hydrogen bond in AAs
12
2.4.1 Group 1: AA with R = H i.e. (Gly)
Gly is the simplest of all AA where the side chain is hydrogen. Due to its
simplicity, the structure of Gly has been studied extensively to build a general framework
on understanding the structure of other complicated AAs.11,12,19,23,24,42,44-54 Gly has three
internal rotational degrees of freedom: the rotation of the hydroxy group around the C1-O
bond (α), the rotation around the Cα-C1 bond (β), and the rotation around Cα-N bond (γ)
as shown in Scheme 2.2.
The interactions shown in Figure 2.2 can be attributed to the rotation around C1-O
and Cα-C1 bonds. For C1-O bond rotation, conformations with O=C-O-H dihedral angles
closer to 0° ( also called cis) or 180° (also called trans) are significant since they favor
type 1 and type 2 hydrogen bonding shown in Figure 2.2, respectively. For Cα-C1 bond
rotation, conformations with N- Cα-C1-O dihedral angles closer to 0° or 180° are
significant since they favor type 4 and type 3 hydrogen bonding shown in Figure 2.2,
respectively.
The rotation around N-Cα determines the number of NH hydrogen involved in
type 3 or type 4 hydrogen bonding, which is defined by < Lp-N-Cα-C1 dihedral angle,
where Lp is the lone pair of electrons in nitrogen. When this dihedral angle is 180° (anti
or a), both hydrogens of amine can hydrogen bond whereas when this dihedral angle is
Scheme 2.2
13
+60° (gauche+ or g+) or -60° (gauche- or g-) only one hydrogen of amine can hydrogen
bond, Scheme 2.3.
The five most stable conformations of Gly are shown in Scheme 2.4.
9,12,41,43,44,46,49,55 All calculations predicts conformation Ia, conformation with type I
(Figure 2.2) hydrogen bonding and anti orientation of < Lp-N-Cα-C1 (Scheme 2.6), as the
most stable form by ~0.5kcal/mol than the next closest conformation. However, the
stability of the other conformations depends on the level of theory and the basis set used
in the calculations. The comparison of the relative energies calculated at the HF and MP2
levels shows the former method overestimates the energy gap between GlyIa and the
other conformations. DFT calculations of the structure and relative stabilities of the Gly
conformers produced results very similar to the results of the MP2 method.
Scheme 2.3
14
Scheme 2.4
Types I and III conformations prefer anti (a) configuration of Lp-N-Cα-C1 since
this configuration allows a bifurcated hydrogen bonding between amine and carboxylic
acid. However, type II conformation prefers gauche (g) orientation of Lp-N-Cα-C1 since
this configuration allows the hydrogen bonding between acidic hydrogen and basic amine.
Although the most stable conformation of Gly predicted computationally has been
consistent, the experimentally determined conformation has varied. Brown et al.23 and
Suenram & Lovas43 are first two groups to independently report similar findings on the
experimental results of the conformation of gaseous Gly in 1978. They both detected the
microwave spectrum of gaseous Gly in a specially constructed cell maintained at a
temperature at which Gly has an appreciable vapor pressure (160-200 °C) while
minimizing the effects of decomposition.23,43 Their findings suggest GlyIIg is the most
abundant conformation, which is contradictory to the computational findings.
Furthermore, they were not able to identify any lines attributing to the lowest energy
conformation, GlyIa.
Suenram & Lovas45 further investigated gaseous Gly with a number of
improvements to their spectrometer, which provided improved sensitivity. In this study,
15
they reported the good agreement on the experimental and theoretical rotational constants
for presence of GlyIa and GlyIIg. In addition, GlyIa was detected to be lower in energy,
by 490 cm-1, when compared to GlyIIg.
Although GlyIIg and GlyIIIa are predicted to have similar energies, there is no
sign on the presence of GlyIIIa in microwave spectroscopy experiment. Godfrey and co-
workers41 stated that GlyIIIa species relaxes to the lower energy GlyIa due to rotation
around C-O bond, augmenting the total concentration of GlyIa in those studies. This can
be understood as the relaxation of GlyIIIa to lowest energy conformer, GlyIa, via a low-
barrier pathway. Since no such low-barrier pathway is available for the relaxation of
GlyIIg, this conformation is observed in these experiments 41,55.
The joint analysis of electron diffraction data and rotational constants performed
by Iijima et. al12 concluded GlyIa to be the main (~76 %) conformer and the rest being a
mixture of GlyIIg and GlyIIIa, although there is no substantial experimental evidence.
Similarly, experiments performed using other techniques have claimed the observation of
GlyIIg and GlyIIIa along with GlyIa10. In addition, Balabin38 recently reported the
presence of new conformer, GlyIg, along with GlyIa and GlyIIe, in an experiment
performed with jet-cooled, high sensitive, Raman spectroscopy. This is the only
experimental incident where the presence of GlyIg is reported.
In summary, 75% of gaseous Gly exists as Ia and the rest as a mixture of IIg, IIIa,
and Ig while experiment is performed in between 200-300K. Almost 100% of Gly will
exist as the main conformer GlyIa at extremely low temperatures, as in cool interstellar
space.12
16
2.4.2 Group 2: AA with Aliphatic Side Chains
The five AAs in this category are Ala, Val, Leu, Ile and Pro. As name suggests,
AAs in this category have a hydrophobic aliphatic side chain where R contain just
carbon(s) and hydrogens. As in Gly, the conformational behavior of these AA can be
expected to be primarily governed by the intramolecular hydrogen bonding within the
AA backbone. However, the increase on size of the side chain complicates the
conformational landscape of the molecule considerably, resulting in a greater number of
plausible conformers27. For example, besides the free rotation discussed in Gly, rotation
around other C-C (for example Cβ- Cα) bonds would generate additional conformers
(Scheme 2.5).
Ala is the most studied AA in this group, which is mainly due to its simple
structure with R=Me. The five most stable conformations of free Ala determined
computationally are shown in Scheme 2.6.50,56-59 The experimental studies performed on
gaseous Ala confirmed the presence of only two conformers, Ala1 and Ala2.26,39-41,60,61
The failure to observe other low-energy conformers given by electronic structure
computations is not surprising since similar results were observed for Gly, which is
attributed to vibrational relaxation.
Scheme 2.5
17
Although the methyl group of Ala has no effect on the lower energy configuration
when compared to Gly, the presence of a bulky isopropyl group in Val (R = CH (CH3)2)
could generate a much steric demand, which could affect the conformation of the AA
back bone of the molecule.16 Theoretical calculations predict three unstrained staggered
orientations of the isopropyl group which are unique structures with different energies.
Considering rotations around Cβ-Cα bond, three unstrained staggered rotamers are
present in Val (Scheme 2.5).
18
The five most stable conformers of a free Val determined computationally are
shown in Scheme 2.9.10,16 The conformational behavior of Val is in closest agreement
with smaller aliphatic neutral α-AAs like Gly and Ala. Affixing an isopropyl side chain to
the α-AA backbone does not alter the relative energies of the lowest energy conformers.
This observation indicates the relative stabilities of the AA skeleton are controlled by
Scheme 2.6
19
intramolecular hydrogen bonding.16 The experimental finding is also comparable to Gly
and Ala. Only Val1 and Val2 are observed in experiments, whereas the absence of other
conformers can be attributed to facile relaxation to lower energy conformers due to
rotation around the C-C bonds.16
As the chain length of side chain grows, the number of C-C bond rotations to
consider increases as well. In the case of Ile and Leu, configuration associated with Cγ-
Cβ rotations as well as rotations in Val need to be considered. Due to this, the number of
possibly lower energy conformers appears in the PES of Leu and Ile will increase by a
factor of 3 compared to Val. The five lowest energy conformers of Leu and Ileu
determined computationally are shown in Scheme 2.6 as rotation around Cγ-Cβ bond. As
for smaller aliphatic AAs, the conformational behavior of Ile and Leu is primarily
governed by the intramolecular hydrogen bonding within the AA backbone. However,
the side chain orientation for the most stable conformation predicted computationally is
the same in Val and Ile but some differences appear on Leu. This can be attributed to a
minimization of steric repulsions within the side chain and the AA backbone since both
Val and Ile have two aliphatic groups connected to the beta carbon.28
Type I and type II configurations (Figure 2.2) are present in experimentally
studies of Leu and Ile with conformation with type I configuration being more stable than
that with type II configuration. This is consistent with the other aforementioned AAs. The
conformation with type I configuration is computationally predicted to be the lowest
energy conformation and is always the most abundant species in experimental results.
Moreover, the conformations with the type II configuration observed experimentally have
the same configuration due to rotation around Cβ-Cα and Cγ-Cβ bonds as for the lowest
20
energy conformation with type I configuration. Even though experimental and
computational results show these similarities for AA’s with aliphatic side chains, it is
different in the case for Leu. Leu2 conformation is computationally predicted to be the
most stable conformer with type II configuration, but Leu3 is observed experimentally. It
is unclear why Leu3 is observed despite its higher energy, but it is clear that in
experimental studies, the same configuration (due to rotation around Cβ-Cα and Cγ-Cβ
bond) as in lowest energy conformation with type I configuration is preferred for
conformation with type II configuration.27,28
Pro is the only α-AA containing a secondary amino group as part of a flexible
five-membered ring. Pro is predicted to have lower energy when the five membered
pyrrolidine ring adopts an envelope conformation.31,42 The envelope conformation of the
ring can be endo-like (en) and exo-like (ex) with respect to the carboxy moiety. All three
types (I, II and III) of hydrogen bonding are possible in Pro but there is only one N-H
hydrogen involving in the formation of intramolecular hydrogen bond with lone pairs of
carboxylic acid oxygen’s. The combination of en and ex with I, II and III give total of six
possible conformations of Pro (only 5 of them is shown in Scheme 2.9). In fact, there are
six lowest energy conformations of Pro and all of these configurations can be
independently detected in gaseous Pro experiment. Unlike the type I configuration
observed in lowest energy conformation of aforementioned aliphatic AAs, Pro exhibits
type II configuration as primary interactions on controlling its stability. As previously
mentioned, the most stable conformer with type I configuration of other AAs has
bifurcated H-bonding of NH2 with O=C, which is not possible in Pro, but this does not
explain variation in inversion of stability of I and II, as in the case of Pro. A comparison
21
can be drawn between Gly and Pro. The N-methylated Gly and N,N-dimethylated Gly
have same conformation preference as in Gly. However, the findings with Pro can be
attributed to a consequence of the torsional restrictions imposed by the five-membered
pyrrolidine ring inverting the stability. 10,31,42,62,63
2.4.3 Group 3: AAs with Oxygen or Sulfur on Side Chain
The four AAs in this category are Ser, Thr, Cys and Met. As discussed in previous
sections, the conformations of AA with non-polar side chains is mainly controlled by the
stabilization due to intramolecular hydrogen bonding between amino and carboxylic acid
groups present in the AA’s backbone. However, due to the polar side chains, the AAs in
this group have a new set of interactions present in them. Hydrogen on S-H or O-H can
interact with lone pair of nitrogen or oxygen of the backbone (e.g. S-H…O-H or
S…H…O=C), except for Met because it lacks a hydrogen on the sulfur. In addition, the
hydrogen on the acid and amine can interact with the loan pair of sulfur or oxygen of side
chain (e.g. O-H...S or N-H…S). These interactions will dramatically increase the number
of low-energy conformers. For example, there exists 51 conformational minima in PES of
Ser.64
The five low energy conformers of Ser, Thr, Cys and Met are shown in Scheme
2.7. It can be noted the low-energy conformations are mainly controlled by the
intramolecular hydrogen bonds established between the three polar groups (COOH, NH2
and S/O). In the case of Ser and Thr, the additional H-bonding between hydroxyl H and
amino group stabilizes the type I configuration. The carboxylic acid group stabilizes type
II and type III configurations. In the case of Cys, both type I and type II configurations
22
with preferred hydrogen bonding between the carboxylic acid and thiol. In addition, the
most stable conformer in Ser, Thr and Met have type I configuration whereas Cys has
type II configuration.
Microwave spectroscopy studies have detected six conformers of Ser56,64, six
conformers of Cys29,56, and seven conformations of Thr14, which is significantly more
than observed for AA’s with aliphatic side chains. Furthermore, conformations with type
III configuration not detected for previously discussed AA’s have been observed for these
AAs. The lack of evidence of type III configurations in experimental studies have been
explained based on its relaxation to lower energy conformer of type I configuration due
to the low-energy barrier. However, the conformers with type III configuration observed
in case of Ser, Cys and Thr have the lowest energy in their family (compared to type I
configuration when every other dihedral angles are kept constant). In addition, the
relaxation from type III configuration to type I configuration has a higher energy barrier,
which hinders the relaxation. For example, the energy barrier for relaxation of Cys4 to
Cys1 is calculated to be approximately 700 cm-1. Hence, it is possible to detect
conformation with type III configuration for this group of AAs. The results obtained from
experimental studies on gaseous Met22,42,63 are on conformations situated more than 10
kJ/mol above the most stable conformers according to the predicted PES. Therefore, the
experimental detection of Met is avoided.10
23
23
Sch
eme
2.7
24
2.4.4 Group 4: AAs with Amine Substituted Side Chain
The two AAs in this category are Lys and Arg. Due to their polarity, several low-
energy conformer of these AA can be expected since new intramolecular hydrogen bonds
are possible. In addition, the long flexible hydrocarbon chain can bend to favor these
interactions.
There are five types of hydrogen-bonding configurations present in these AA:
(a) NH2…OCOH…NH2, (b) COOH…NH2…NH2, (c) H2N…HOCO…H2N,
(d) COOH…NH2 and (e) HOCO…H2N. However, due to the free rotation around all C-C
bonds, only the first two types are present for the five low energy conformers of Lys and
Arg shown in Scheme 2.8. It can be noted the lowest energy conformer of both Lys and
Arg has type II configuration, where –COOH is in the trans orientation. The first three
Scheme 2.8
25
lowest energy conformers of Lys have NH2…OCOH…NH2 and 4th and 5th conformers
have COOH…NH2…NH2 hydrogen bonding configurations where first NH2 is from the
AA backbone and second amine is from the side chain in both cases. In case of Arg, the
1st and 4th conformers have H2N…HOCO…H2N. The 2nd, 3rd and 5th conformers have
NH2…OCOH…N where the first NH2 is from AA backbone and second one is from side
chain in both cases and N is a nitrogen lacking hydrogen.65
2.4.5 Group 5: AAs with Carbonyl Substituted Side Chain
The four AAs in this category are Asp, Asn, Gln and Glu. Unlike previous
sections where polar side chains would interact with the polar backbone, AAs in this
section have a capacity to interact and form H-bonding within the side chain, which
increases the possible number of low-energy conformers on the PES.
The five low energy conformations of Asp, Glu, Asn and Gln are shown in
Scheme 2.9. All conformations of Asp and Glu have cis orientation of the COOH side
chain due to a 20 kcal/mol stabilization energy compared to the trans orientation. In
addition, all conformations have type II configuration of AA backbone and additional
hydrogen bonding with the side chain or they have type I configuration of AA backbone
for further stability.33 Since Asn and Gln have amine side chains, one hydrogen of amine
NH2 side chain form H-bond with acid of AA backbone and another with C=O of amide
in all cases. Interestingly, the most stable conformation of Asn adopts a ring like
structures due to presence of continuous hydrogen bonding.
26
NH
H
H
OO
H
Asp
NH
H
H
OO
H
H H
HHNH
H
H
OOH
HH
O OO
H
O
H
O
O H
NH
H H
OOH
HH
O
HO
NH
H H
OO
H
H
HOO
H
H
H
H
H
N
OO
H
O
OH
H
H
H
HH
H
N
O
O
H
O H
O
H
H
H
H
HH
N
O
O
H
H
H
OO
H
H
H
H
H
N
O
O H
OO
H
H
H
Glu
H
H
H
H
N
O
O H
H
H
O
O
H
Asn
NH
H
H
OO
H
HH
O
N H
NH
H
H
OOH
HH
NH
O
H
NH
H H
OOH
HH
N
HOH
NH
H H
OO
H
H
N HO
H
H
H
NH
H H
OOH
H
H
N
HOH
H
H
H
H
N
OO
H
O
NH
H
H
H
H
HH
O
H
H
H
H
N H
ON
H
O
Gln
H
NH H
NH
O
OH
H
H
O
H
H
H
HH
H
NH
O
O H
H
O
NH
HH
HH
H
NH
O
O H
H
ON
HH
1 2 3 4 5
2.4.6 Group 6: AAs with Aromatic Side Chain
The four AAs in this category are Tyr, Trp, Phe and His. These aromatic AAs
have ultraviolet radiation absorptions with large extinction coefficients. This feature is
often used as an ultraviolet marker for detection of these AAs, either separately, or
incorporated into proteins and enzymes, via ultraviolet spectrophotometry.
Scheme 2.9
27
The presence of an electron rich aromatic ring in the side chain stabilizes the
structure by utilizing interactions between the polarized hydrogen and the pi-electron.
The five low energy conformers of this group are shown in Scheme 2.10.10,66-71 The most
stable structure has trans COOH with O-H … NH2 hydrogen bond, which is further
stabilized by interaction of NH2 hydrogen with the pi-system of the aromatic ring. This
stability is consistent for all AAs in this group.
The conformation of Phe is of great interest to our research group since we have
stated to study charge-remote fragmentation of Phe in MS.72 The formation preferences
of negatively and positively charged Phe, PheSO3 and PheNMe3+, are shown in Figures
5.1 and 6.2 of this thesis, respectively. The low energy conformations of PheSO3 are
similar to Phe where Type II conformation is preferred whereas the low energy
conformations of PheNMe3+ prefer Types I and III conformations.
This concludes the structure of canonical AAs in gas phase. The next section is on
stabilization of zwitterionic AA in gas phase.
Scheme 2.10
28
2.5 Stabilization of Zwitterionic AAs in Gas Phase
Zwiterionic isomers of AA have been proved to be unstable in the gas phase.
However, they can be stabilized in gas-phase clusters through solvation with water17,73,
methanol17, metal cation17,53, zeolite18, chloride17 and ammonium ion19. Because the
zwitterion formation is important for the structure, function and activity of biological
systems involving peptides, proteins and nucleic acids, knowing what conditions stabilize
the zwitterion is essential. Several theoretical and experimental studies are performed to
understand the effects of solvation on the relative stability of canonical and zwitterionic
conformers.
The recent theoretical study by Pathak17 predicts two solvent (water and
methanol) molecules or a single ion can sufficiently stabilize the zwitterionic form of all
α-AAs. A combination of experimental and theoretical study on Trp by Polfer et.al73
reported that water preferentially binds on the carboxylic acid group and the water
molecules strongly affects the relative energetics of different structural motifs.
One of the very first ab initio studies examining how the successive addition of
water molecule affects the chemical dynamic of ZN equilibrium of the AA Gly
performed by Jensen and Gordon.8 They studied the effect of solvation (by 1 and 2 water
molecules) on the transition state energy and enthalpy of ZN proton transfer (PT)
reactions. Even though the zwitterionic form of Gly does not exist in the gas phase,
calculations were performed on the isolated structure because it provides valuable
information about the intrinsic chemistry of this species necessary for a direct evaluation
of the solvent effect. They discovered 1, 2 and 3 unique zwitterionic minima in the PES
of bare, mono-hydrated and di-hydrated Gly, respectively.
29
Since the only interaction present in Z0 (Gly Zwitterion with 0 water molecule) is
intramolecular hydrogen bonding, the proton transfer has to occur internally. The PT
reaction, Z0 N0, is calculated to be exothermic by 17 kcal/mol and the transition state
for this reaction is only 0.02 kcal/mol higher than Z0, indicating Z0 readily collapses to
N0. The destabilization of Z in the gas-phase has been attributed to the shorter (1.55 Å)
intramolecular hydrogen bond distance in Z0 than in N0 (1.97 Å) and to the fact the
proton affinity of COO anion is higher (by 129 kcal/mol) than the NH2.
The mono- and di-hydrated Gly can have either intra or intermolecular hydrogen
bonding which can assist the PT reaction. The Z1 conformers have a water molecule
bridging the COO and NH3+ groups. Intramolecular and intermolecular PT reactions are
calculated to be exothermic by 11.8 kcal/mol and 10.4 kcal/mol, respectively, with no
barrier for both mechanisms. It is apparent one water molecule is not enough to stabilize
the zwitterionic form of Gly. However, for the PT reaction of Z2 N2 is calculated to be
exothermic by 6.3 kcal/mol with a positive barrier of 1.9 kcal/mol. Furthermore, Z2
structure with intermolecular hydrogen bonding, and not with only intramolecular H-
bond, is the stable one. Hence, the study concludes two water molecules can stabilize Gly
zwitterionic structure in the gas-phase and the electrostatic interaction (H-bonding)
between water and the AA is essential for the stability of this structure.
Several theoretical and experimental studies have been performed since then to
determine how many water molecules are necessary to stabilize the Z conformer of
AAs.8,21,51,59,65,73-75 The zwitterionic structures of Arg, Asp acid, Asp, Cys, Phe, Ser, Thr
and Tyr can be stabilized by as few as one water molecule.17 The common feature among
them, with the exception of Phe, is a polar side chain. Interestingly, the other AAs with
30
polar side chains such as Glu, Gln and Lys, and with nonpolar side chain such as Ala, Gly,
His, Ile, Leu, Met, Pro, Typ and Val require two molecules of water to stabilize the
zwitterionic structure.17 The zwitterionic forms are stabilized by the formation of
hydrogen bonding interactions with water molecules. In general, the negative end of the
solvent dipole orients toward the basic site of AA and the positive end towards the acid
site of AA.17
Both theoretical and experimental studies carried out to examine the relative
stability of the canonical and zwitterionic forms as a function of the number of
microsolvating water molecules indicated that the effects of microsolvating water
molecules depend highly on the structure of AAs. Although most AAs require 4-5 water
to stabilize their zwitterionic conformers molecules those with strongly basic side chains
such as Arg and Lys may need fewer water molecules. 74,76Val is at the opposite side to
these latter AAs in the effects of side chain.20,53 The isopropyl side chain is highly
nonpolar, and is neither acidic nor basic. Val is a normal AA in its behavior towards the
effects of microsolvating water molecules, in contrast to other AAs such as Arg and Lys
whose zwitterions were predicted to become stable under the influence of fewer number
of water molecules due to side chains of strong basicity.20
In addition, calculations for the Arg-H2O system predicts the zwitterionic Arg be
thermodynamically more stable than the canonical form in the gas phase under the
influence of a single water molecule because of the strongly basic guanidine side chain.74
Canonical conformers of Arg-H2O are found to isomerize to the zwitterionic forms via a
small barrier (∼6 kcal/mol).74 In contrast to other AAs for which more water molecules
31
are required to stabilize the zwitterion, in case of Arg three water molecules are enough
because of the strongly basic side chain.
2.6 Conclusion This chapter summarizes conformational landscape of the 20 α-amino acids in gas
phase. The intramolecular hydrogen bonding mainly stabilizes the conformation of
canonical amino acid in gas phase. Zwiterionic isomers of AA have been proved to be
unstable in the gas phase, which exist predominately in the condensed phase. However,
they can be stabilized in gas-phase clusters through solvation with water, methanol, metal
cation, zeolite, chloride and ammonium anion.
32
2.6 References (1) Albrecht, G.; Corey, R. J. Am. Chem. Soc. 1939, 61, 1087.
(2) Marsh, R. Acta Crystallogr. 1958, 11, 654.
(3) Levy, H.; Corey, R. J. Am. Chem. Soc. 1941, 63, 2095.
(4) Donohue, J. J. Am. Chem. Soc. 1950, 72, 949.
(5) Gaffney, J.; Pierce, R.; Friedman, L. J. Am. Chem. Soc. 1977, 99, 4293.
(6) Cohn, E.; McMeekin, T.; Edsall, J.; Blanchard, M. J. Am. Chem. Soc. 1934, 56, 784.
(7) Ellzy, M.; Jensen, J.; Hameka, H.; Kay, J. Spectrochim. Acta, Part A 2003, 59, 2619.
(8) Jensen, J.; Gordon, M. J. Am. Chem. Soc. 1995, 117, 8159.
(9) Jensen, J.; MS, G. J. Am. Chem. Soc. 1991, 113, 7917.
(10) Bouchoux, G. Mass Spectrom. Rev. 2012, 31, 391.
(11) Lago, A.; Coutinho, L.; Marinho, R.; de Brito, A.; de Souza, G. Chem. Phys. 2004, 307, 9.
(12) Iijima, K.; Tanaka, K.; Onuma, S. J. Mol. Struct. 1991, 246, 257.
(13) Jarrold, M. Ann. Rev.Phys. Chem. 2000, 51, 179.
(14) Alonso, J.; Perez, C.; Sanz, M.; Lopez, J.; Blanco, S. Phys. Chem. Chem. Phys. 2009, 11, 617.
(15) Tia, M.; de Miranda, B.; Daly, S.; Gaie-Levrel, F.; Garcia, G.; Nahon, L.; Powis, I. J. Phys. Chem. A 2014, 118, 2765.
(16) Lesarri, A.; Cocinero, E.; Lopez, J.; Alonso, J. Angew. Chem. Int. Ed. 2004, 43, 605.
(17) Pathak, A. Chem. Phys. Lett. 2014, 610, 345.
(18) Yang, G.; Zhou, L.; Liu, C. J. Phys. Chem. B 2009, 113, 10399.
(19) Wu, R.; McMahon, T. J. Am. Chem. Soc. 2008, 130, 3065.
(20) Kim, J.; Won, G.; Lee, S. Bull. Korean Chem. Soc. 2012, 33, 3797.
33
(21) Vaquero, V.; Sanz, M.; Mata, I.; Cabezas, C.; Lopez, J.; Alonso, J. J. Phys. Chem. A 2014, 118, 2584.
(22) Linder, R.; Seefeld, K.; Vavra, A.; Kleinermanns, K. Chem. Phys. Lett. 2008, 453, 1.
(23) Brown, R.; Godfrey, P.; Storey, J.; Bassez, M. J. Chem. Soc., Chem. Commun. 1978, 547.
(24) McGlone, S.; Elmes, P.; Brown, R.; Godfrey, P. J. Mol. Struct. 1999, 485, 225.
(25) Linder, R.; Nispel, M.; Haber, T.; Kleinermanns, K. Chem. Phys. Lett. 2005, 409, 260.
(26) Blanco, S.; Lesarri, A.; Lopez, J.; Alonso, J. J. Am. Chem. Soc. 2004, 126, 11675.
(27) Lesarri, A.; Sanchez, R.; Cocinero, E.; Lopez, J.; Alonso, J. J. Am. Chem. Soc. 2005, 127, 12952.
(28) Cocinero, E.; Lesarri, A.; Grabow, J.; Lopez, J.; Alonso, J. ChemPhysChem 2007, 8, 599.
(29) Sanz, M.; Blanco, S.; Lopez, J.; Alonso, J. Angew. Chem. Int. Ed. 2008, 47, 6216.
(30) Cabezas, C.; Varela, M.; Pena, I.; Mata, S.; Lopez, J.; Alonso, J. Chem. Commun. 2012, 48, 5934.
(31) Lesarri, A.; Mata, S.; Cocinero, E.; Blanco, S.; Lopez, J.; Alonso, J. Angew. Chem. Int. Ed. 2002, 41, 4673.
(32) Mata, S.; Vaquero, V.; Cabezas, C.; Pena, I.; Perez, C.; Lopez, J.; Alonso, J. Phys. Chem. Chem. Phys. 2009, 11, 4141.
(33) Sanz, M.; Lopez, J.; Alonso, J. Phys Chem Chem Phys 2010, 12, 3573.
(34) Pena, I.; Sanz, M.; Lopez, J.; Alonso, J. J. Am. Chem. Soc. 2012, 134, 2305.
(35) Bermudez, C.; Mata, S.; Cabezas, C.; Alonso, J. Angew. Chem. Int. Ed. 2014, 53, 11015.
(36) Sanz, M.; Cabezas, C.; Mata, S.; Alonso, J. J. Chem. Phys. 2014, 140.
(37) Balabin, R. J. Chem. Phys. Letters 2010, 1, 20.
34
(38) Balabin, R. Phys. Chem. Chem. Phys. 2010, 12, 5980.
(39) Jaeger, H.; Schaefer, H.; Demaison, J.; Csaszar, A.; Allen, W. J. Chem. Theory Comput. 2010, 6, 3066.
(40) Godfrey, P.; Firth, S.; Hatherley, L.; Brown, R.; Pierlot, A. J. Am. Chem. Soc. 1993, 115, 9687.
(41) Godfrey, P.; Brown, R.; Rodgers, F. J. Mol. Struct. 1996, 376, 65.
(42) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K.; Carravetta, V. J. Electron. Spectrosc. Relat. Phenom. 2007, 155, 47.
(43) Suenram, R.; Lovas, F. J. Mol. Spectrosc. 1978, 72, 372.
(44) Schafer, L.; Sellers, H.; Lovas, F.; Suenram, R. J. Am. Chem. Soc. 1980, 102, 6566.
(45) Suenram, R.; Lovas, F. J. Am. Chem. Soc. 1980, 102, 7180.
(46) Tse, Y.; Newton, M.; Vishveshwara, S.; Pople, J. J. Am. Chem. Soc. 1978, 100, 4329.
(47) Brown, R.; Godfrey, P.; Storey, J.; Bassez, M.; Robinson, B.; Batchelor, R.; Mcculloch, M.; Rydbeck, O.; Hjalmarson, A. Mon. Not. R. Astron. Soc. 1979, 186, P5.
(48) Csaszar, A. J. Am. Chem. Soc. 1992, 114, 9568.
(49) Hu, C.; Shen, M.; Schaefer, H. J. Am. Chem. Soc. 1993, 115, 2923.
(50) Csaszar, A. J. Mol. Struct. 1995, 346, 141.
(51) Alonso, J.; Pena, I.; Sanz, M.; Vaquero, V.; Mata, S.; Cabezas, C.; Lopez, J. Chem. Commun. 2013, 49, 3443.
(52) Balta, B.; Basma, M.; Aviyente, V.; Zhu, C.; Lifshitz, C. Int. J. Mass
spectrum. 2000, 201, 69.
(53) Dunbar, R.; Polfer, N.; Oomens, J. J. Am. Chem. Soc. 2007, 129, 14562.
(54) Kasalova, V.; Allen, W.; Schaefer, H.; Czinki, E.; Csaszar, A. J. Comput. Chem. 2007, 28, 1373.
(55) Godfrey, P.; Brown, R. J. Am. Chem. Soc. 1995, 117, 2019.
(56) Gronert, S.; O'Hair, R. J. Am. Chem. Soc. 1995, 117, 2071.
35
(57) Cao, M.; Newton, S.; Pranata, J.; Schafer, L. THEOCHEM 1995, 332, 251.
(58) Csaszar, A. J. Phys. Chem. 1996, 100, 3541.
(59) Mullin, J.; Gordon, M. J. Phys. Chem. B 2009, 113, 8657.
(60) Stepanian, S.; Reva, I.; Radchenko, E.; Adamowicz, L. J. Phys. Chem. A 1998, 102, 4623.
(61) Hirata, Y.; Kubota, S.; Watanabe, S.; Momose, T.; Kawaguchi, K. J. Mol. Spectrosc. 2008, 251, 314.
(62) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K.; Carravetta, V. Chem. Phys. Lett. 2007, 442, 429.
(63) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K.; Carravetta, V. J. Phys. Chem. A 2007, 111, 10998.
(64) Blanco, S.; Sanz, M.; Lopez, J.; Alonso, J. Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 20183.
(65) Leng, Y.; Zhang, M.; Song, C.; Chen, M.; Lin, Z. THEOCHEM 2008, 858, 52.
(66) Kaczor, A.; Reva, I.; Proniewicz, L.; Fausto, R. Journal of Physical Chemistry a 2006, 110, 2360.
(67) von Helden, G.; Compagnon, I.; Blom, M.; Frankowski, M.; Erlekam, U.; Oomens, J.; Brauer, B.; Gerber, R.; Meijer, G. Physical Chemistry Chemical Physics 2008, 10, 1248.
(68) Huang, Z.; Yu, W.; Lin, Z. Journal of Molecular Structure-Theochem 2006, 801, 7.
(69) Huang, Z.; Yu, W.; Lin, Z. Journal of Molecular Structure-Theochem 2006, 758, 195.
(70) Snoek, L.; Robertson, E.; Kroemer, R.; Simons, J. Chemical Physics Letters 2000, 321, 49.
(71) Perez, C.; Mata, S.; Blanco, S.; Lopez, J.; Alonso, J. Journal of Physical Chemistry a 2011, 115, 9653.
(72) Koirala, D.; Kodithuwakkuge, S.; Wenthold, P. J. Phys. Org. Chem. 2015, 28, 635.
36
(73) Blom, M.; Compagnon, I.; Polfer, N.; von Helden, G.; Meijer, G.; Suhai, S.; Paizs, B.; Oomens, J. J. Phys. Chem. A 2007, 111, 7309.
(74) Im, S.; Jang, S.; Lee, S.; Lee, Y.; Kim, B. J. Phys. Chem. A 2008, 112, 9767.
(75) Tian, S.; Sun, X.; Cao, R.; Yang, J. J. of Physical Chemistry a 2009, 113, 480.
(76) Hwang, T.; Eom, G.; Choi, M.; Jang, S.; Kim, J.; Lee, S.; Lee, Y.; Kim, B. J. Phys. Chem. B 2011, 115, 10147.
37
CHAPTER THREE: EXPERIMENTAL APPROACHES
3.1 Introduction
A general overview of the experimental setup and theoretical procedures used in
this thesis are presented in this chapter. Experimental results presented throughout this
thesis are obtained from two mass spectrometry (MS) instruments; namely, flowing-
afterglow triple-quadrupole and LCQ-Deca MS. The flowing afterglow,1 Figure 3.1, is a
homebuilt instrument and LCQ-Deca is a commercial instrument of Thermo Scientific.2
Both of them contain an ion source, mass analyzer and detector, which are common
features of all MS instruments.1,3-7 Chemical compounds are first ionized in the ion
source region. These ions are then transferred to the low-pressure chamber, containing
the mass analyzer, where they are manipulated and discriminated based on mass/charge
(m/z) ratios by electro-magnetic fields. Finally, they are detected and the generated signal
is processed to produce a mass spectrum.6 Our goal in this chapter is to address how these
features are adopted in each of our MS and to specify the methods used to generate data
presented in this thesis. More detailed descriptions of the flowing afterglow1,5 and LCQ-
Deca2 have been provided elsewhere.
38
3.2 The Flowing-Afterglow Triple-Quadrupole Mass Spectrometer
All experiments described in chapter four of this thesis were conducted using a
flowing afterglow triple-quadrupole mass spectrometer. This section is divided in three
sections; describing the ion source, the mass analyzer and the detector implemented in the
flowing afterglow triple-quadrupole MS.
Figure 3.1. The flowing-afterglow triple-quadrupole mass spectrometer.
3.2.1 The Ion Source
The flowing afterglow MS utilizes electron impact ionization method to generate
ions of interest. Historically, electron impact ionization, also known as electron ionization
(EI), was the most commonly used ionization method to create gas-phase ions for a wide
range of organic molecules.6,7 When an electron hits a neutral molecule, it may eject an
39
electron to produce positive ions or combine to form negative ions. Negative ions may
also be formed by dissociative attachment.1
This instrument uses an electron gun consisting of a rhenium metal filament to
emit free electrons when resistively heated. The electrons formed in an ion source are
attracted toward the metal mesh where 70 eV of biased potential is applied. In addition,
there is a continuous flow of dry, helium, carrier gas through an inlet situated adjacent to
the electron gun (Figure 3.1). The helium gas has two purposes: 1) to thermalize the
electrons and 2) to carry electrons downstream for electron ionization.1,5
Ions are synthesized in a one-meter-long flow tube in the gas phase (Figure 3.1).
The mixture of He and electrons are pumped downstream of the flow tube due to a
pressure gradient created by a roots blower vacuum pump. There are six inlet ports on the
flow tube through which vapors of compounds are introduced into the tube and ions are
generated by electron impact ionization. In addition, gas-phase ion-molecule reactions of
the ions residing in flowing helium can be studied by adding reagents like O2, CO2, NO
and CS2 from ports located further downstream on the flow tube.8,9 Furthermore, the flow
tube possesses kinetic rings to introduce a neutral reagent vapor at a calibrated flow rate
and then measuring the extent of reaction.
EI can be applied to volatile samples only because the sample has to be in the gas
phase before electron can ionize it. EI is an endothermic process and if the internal
energy of the compound is less than the energy deposited, the molecule fragments, which
obscures the m/z information of the analytes. Due to this reason, it is categorized as a
hard ionization method.6,7,10 The problem of fragmentation has been overcome by
chemical ionization (CI), a method using gas-phase chemical reactions to produce ions
40
with little to no fragmentation.8,11 All the ions studied in Chapter four are generated by EI.
However, the flowing afterglows the capability for CI is also limited to volatile and
thermally stable organic compounds, and needs to be performed in a low-pressure
atmosphere.
The first step in CI is to form reagent ions by EI, which are then reacted with a
molecule to produce ions of interest. The most useful reagent ions in a negative ion mode
include F- and OH-, which is mainly because these ions are very basic and can easily
deprotonate analytes to form anions of interest. The reagent ions, F- can be created by EI
of fluorine gas and OH- from a 2:1 mixture of methane and nitrous oxide, respectively
(equations 3.1 and 3.2).
(3.1)
(3.2)
3.2.2 The Mass Analyzer
The next region of the flowing afterglow instrument is a triple quadrupole mass
analyzer, which is differentially pumped at 10-6-10-7 Torr by two diffusion pumps. This
section of the instrument is separated from the flow tube by a molybdenum disk held at a
~1-2 V attracting potential. A small amount of gaseous ions produced in the flow tube are
sampled through a 1 mm orifice in a nosecone and are focused to the mass analyzer
41
region by a set of four lenses. The analyzer in the flowing afterglow is a beam type,
where the ions enter and pass through the analyzing field to the detector as a beam,
consisting of three axially aligned quadrupoles. In addition, there are lenses before
(entrance lens) and after (exit lens) the second quadrupole to help focus and guide ions in
and out. The first quadrupole (Q1) can be used to scan a range of m/z (scanning mode) or
to isolate a single m/z (single ion mode). When Q1 is used in scanning mode, the second
(q2) and third (Q3) quadrupoles are used to transfer all ions with no m/z discrimination.
If Q1 is used in the single ion mode, then the ions selected in Q1 can be fragmented in q2
by colliding it with an inert target gas (Ar) or reacted with reagent gases. The first
process is called collision-induced dissociation and the second process is referred to as q2
reactivity. During CID, Ar is dispenses at a pressure of ~150 uTorr with ~10 V of q2 pole
offset. Whereas, q2 reactivity is performed by dispensing reagent gases at a pressure of
~100 uTorr with q2 pole offset close to 0 V to prevent fragmentation. In these processes,
Q3 is used as the mass analyzer. The fragments formed from CID experiments help to
interpret structure of a molecule and q2 reactivity is used to verify the reaction products
observed in the flow tube.
3.2.3 Ion Detection Region
The detector in the flowing afterglow consists of a conversion dynode and an
electron multiplier. The negative ions coming out of Q3 are attracted by the conversion
dynode’s accelerating potential of +1500 V. These ions strike the inner walls of the
electron multiplier and eject electrons. The ejected electrons travel down the funnel
shaped electron multiplier and produce a cascade of electrons resulting in a measurable
42
current proportional to the intensity of the ion. Finally, the signal is collected using the
Q50 software where a mass spectrum is obtained.
3.3 The LCQ-Deca Mass Spectrometer
All experiments described in chapter four, five and six of this thesis were
conducted on LCQ-Deca Mass Spectrometer. This section is divided in three sections;
describing the ion source, the mass analyzer and the detector implemented in the LCQ-
Deca.
3.3.1 The Ion source
The LCQ-Deca uses electrospray ionization (ESI) to produce ions of interest. ESI
is one of the several methods, which ionizes non-volatile and thermally instable
compounds. It also creates little to no fragmentation and is categorized as a soft
ionization method.4,6,7,12-14 However, this method requires compounds to be soluble in
polar solvents like water, methanol, and acetonitrile.14 Generally, a dilute analyte solution
is injected through a hypodermic needle or stainless steel capillary. A very high voltage
(2–6 kV) is applied to the tip of the metal capillary, which causes the dispersion of the
sample solution into an aerosol of highly charged electrospray (ES) droplets. The droplets
are nebulized with a nonreactive gas (usually dry N2) to create a fine spray in which
solvent evaporation occurs as the droplets traverse the atmospheric interface, introducing
molecular ions into the analyzer.4,13 However, the actual mechanism for generating gas-
phase ions via ESI differs depending upon the analyte in question.
43
Ions in the LCQ-Deca are transferred from solution to the gas phase through an
ESI probe. The ESI probe includes sample tube, spray needle, sheath and auxiliary gas
inlets, and nozzle. Solution enters the ESI probe through the sample tube into the spray
needle. The sample tube could be a stainless steel or a fused-silica tube. In our instrument,
0.1 mm ID X 0.19 mm OD fused-silica sample tube is used. The sample tube is located
inside the metal spray needle, where a large negative or positive voltage (typically ±3 to
±5 kV) is applied. This allows the solution to be sprayed into a fine mist of charged
droplets. An ESI nozzle directs the flow of N2, as a sheath and an auxiliary gas, at the
droplets. Sheath gas is an inner coaxial nitrogen gas helping nebulize droplets into a fine
mist as it exits the nozzle. The auxiliary gas is the outer coaxial nitrogen gas assisting the
sheath gas in the nebulization and evaporation of sample solutions. For the experiments
reported in this thesis, the auxiliary gas is not used and a sheath gas flow rate is set in
between 20-30 units of instrumental parameter.
3.3.2 The Mass Analyzer
Located in between the ion source and mass analyzer are a heated capillary, a tube
lens and ion optics. The heated capillary is a heater-embedded metal tube with a hole
bored through the center of its long axis. Ions are drawn into the heated capillary in the
atmospheric pressure region and are transported to the capillary-skimmer region of the
vacuum manifold by a decreasing pressure gradient. Hence, the heated capillary assists in
further desolvating the ions. Ions exiting the heated capillary are focused and accelerated
44
toward the tube lens due to the offset voltage applied on the lens. The tube lens also
serves as a gate to stop the injection of ions into the mass analyzer during ion detection.
Ions from tube lens pass through the skimmer and enter the ion optics region. The
skimmer acts as a vacuum baffle between the higher pressure (1 Torr) and the lower
pressure ion optics regions (10-3 Torr). The ion optics consist a quadrupole, a lens and an
octapole. The quadrupole and octapole act as a transmission device to guide the ions to
the mass analyzer. The lens located in between assists in the focusing ions during the
injection and gating of ions during mass analysis. When a supplemental DC voltage is
added to the overall voltage gradient across the ion path, the non-selective ion
fragmentation occurs, which is called source CID. We have employed source CID in
several occasions to improve the intensity of the secondary ions, which can be studied
later in the mass analyzer.
The mass analyzer in the LCQ-Deca is a quadrupole ion trap, which utilizes a
three-dimensional field to trap ions.6,7,14 The quadrupole ion trap (QIT) is similar to the
quadrupole mass analyzer (QMA) present in flowing afterglow. Whereas a QMA has
electric fields in two dimensions (x and y) and the ions move perpendicular to the field
(the z direction), the QIT has the electric field in all three dimensions, which can result in
ions being trapped in the field.6,7 Ions with a stable trajectory through the quadrupole are
detected in QMA, whereas the ions with unstable trajectories get ejected from the trap
and are detected in QIT.15
The QIT in Deca includes three stainless steel electrodes: two of those are endcap
electrodes, known as the entrance and the exit, the third is a hyperbolic ring situated
centrally between the end-cap electrodes to form a cavity. The two quartz spacer rings
45
serving as electric insulators separate these electrodes. Ions are injected to the mass
analyzer cavity via a small opening in the entrance end cap electrode. Helium is used as a
dampening gas inside the ion trap, due to its ability to cool the ions without inducing
fragmentation. The ions are focused to the axis of the cavity due to dampening and are
trapped due to the RF voltage applied in the ring electrode. A resonance ejection RF
voltage is applied to the endcaps of the mass analyzer to minimize space charge effects in
the mass analyzer. When an ion is to be ejected from the mass analyzer cavity, it is
brought towards the frequency of the resonance ejection RF voltage. The application of
resonance ejection increases resolution because ions of a specific m/z leave the trap in a
more condensed manner.
An ion of interest can be isolated in QIT of LCQ-Deca by ejection of other ions
out of the trap. The ions with different m/z have different oscillatory frequencies in the
ion trap so can be separated. The ion isolation waveform voltage, which consists of a
distribution of resonance frequencies corresponding to unwanted ions, is applied to the
endcaps to eject all ions except those of a selected m/z. Thus isolated ions can be excited
by applying a resonance excitation RF voltage to the endcaps, which creates energetic
collisions of ions with the background helium gas and cause parent ions to fragment into
product ions. However, the resonance excitation RF voltage is not strong enough to eject
an ion from the mass analyzer. Therefore applied voltage can be tuned to get desired
fragmentation. Sequences of isolation followed by fragmentation then isolation again can
be performed for MSn experiment.
46
3.3.3 Ion Detection Region
The detector in the LCQ-Deca consists of a conversion dynode and an electron
multiplier. Ions are attracted toward the conversion dynode due to applied offset voltage
and produce secondary particles when ions hit the surface of the dynode. These
secondary particles are focused and are accelerated by a voltage gradient into the electron
multiplier. Secondary particles from the conversion dynode strike the inner walls of the
electron multiplier and eject electrons. The ejected electrons travel down the funnel
shaped electron multiplier and produce a cascade of electrons. It results in a measurable
current proportional to the intensity of the ion. Finally, the signal is collected using the
Xcalibur data system where a mass spectrum is obtained.
47
3.4 References
(1) Graul, S.; Squires, R. Mass Spectrom. Rev. 1988, 7, 263.
(2) LCQ Deca Mass Spectrometry, Manual
(3) Demartini., D. R. A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses, 2013.
(4) Cooks, R.; Ouyang, Z.; Takats, Z.; Wiseman, J. Science 2006, 311, 1566.
(5) Marinelli, P.; Paulino, J.; Sunderlin, L.; Wenthold, P.; Poutsma, J.; Squires, R. Int. J. Mass Spectrom. Ion Processes 1994, 130, 89.
(6) Maher, S.; Jjunju, F.; Taylor, S. Rev Mod Phys 2015, 87, 113.
(7) Glish, G.; Vachet, R. Nat. Rev. Drug Discov. 2003, 2, 140.
(8) Rau, N.; Welles, E.; Wenthold, P. J. Am. Chem. Soc. 2013, 135, 683.
(9) Koirala, D.; Poole, J.; Wenthold, P. Int. J. Mass spectrom. 2015, 378, 69.
(10) WANNIER, G. Phys. Rev. 1953, 90, 817.
(11) Munson, M.; Field, F. J. Am. Chem. Soc. 1966, 88, 2621.
(12) Takats, Z.; Wiseman, J.; Gologan, B.; Cooks, R. Science 2004, 306, 471.
(13) Banerjee, S.; Mazumdar, S. Int. J. Anal. Chem. 2012.
(14) El-Aneed, A.; Cohen, A.; Banoub, J. Appl. Spectrosc. Rev 2009, 44, 210.
(15) Stafford, G.; Kelley, P.; Syka, J.; Reynolds, W.; Todd, J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85.
48
CHAPTER FOUR: THE FLOWING AFTERGLOW STUDY OF PYRIDINE N-OXIDE NITRENE RADICAL ANIONS
4.1 Introduction
Nitrenes are a fascinating class of electron-deficient and highly reactive
intermediates. They have a nitrogen motif with two bonding and four non–bonding
electrons in valence shell which are isoelectronic to their carbon analogues carbenes.
Nitrenes are nominally sp-hybridized with two electrons in the lower energy sp-orbital as
a pair, whereas the remaining two electrons can be oriented in two available p-orbitals in
four different ways as illustrated in Scheme 4.1. Although isoelectronic, nitrenes and
carbenes show very different reactivity.1-3 For example, both phenylcarbene (PhC) and
phenylnitrene (PhN) can be generated by photolysis of nitrogen containing precursors,
and are generated in the lowest energy singlet state. The initially formed closed–shell
singlet PhC can rapidly undergo intersystem crossing (ISC) to form a ground state triplet
which shows clean bimolecular reactivity. ISC is not favorable for PhN, so PhN
undergoes unimolecular reaction via lowest energy singlet resulting in forming of
polymeric tar when reacted with alkene, alcohol, and aromatic compounds.1,4
49
There are several studies carried out in the reactivity of nitrenes and their study
has led to many important mechanistic ideas, 1,4-6 however, only few studies of nitrene
radical anions have been reported. In addition, many of these studies are focused on the
use of the anions as precursors for photoelectron/photodetachment spectroscopy studies
of the corresponding nitrenes.7-11 Nitrene radical anions are formed when an electron is
added to nitrene. The PhN shown in Figure 4.1 can have two PhN¯ radical anions. Beside
sp-hybridezed electrons, PhN¯ consist of three electrons in two non-bonding molecular
orbitals, an in-plane σ orbitals and a π orbital (Scheme 4.2). The electronic structures of
phenylnitrene anions have been discussed in the context of photoelectron10,11 and
photodetachment7-9 spectroscopy studies, and have been examined by electronic structure
calculations. These studies have shown that the ground state of PhN is π2σ, with the σ2π
state lying approximately 0.5eV higher in energy.
Scheme 4.1
50
Benzoylnitrene radical anion, BzN, has been examined computationally and
experimentally.12-14 Like PhN, the ground state of BzN is predicted to be π2σ, but the
relative energy of the σ2π state (28 kcal/mol) is predicted to be much higher than that in
PhN, reflecting a greater preference for the carboxylate–like anion over having the
charge localized on a nitrogen σ orbital. Chemical reactivity of the BzN is consistent
with the open–shell structure like in PhN, in particular, reaction with NO results in
radical–radical coupling to give a net nitrogen/oxygen exchange, leading to the formation
of benzoate anion (eq 4.1).
(4.1a)
(4.1b)
Scheme 4.2
51
Here, we investigate the reactivity of the 3– and 4–pyridinylnitrene–n–oxide
radical anions, 3PNO and 4PNO respectively.
These are interesting isomers to study because they contain the moieties of two
well characterized compounds: phenylnitrene anion (PN) and pyridine–N–oxide (PO).
PN contains the unusual hypovalent anionic nitrene, whereas PO has a persistent
zwitterionic N–oxide moiety. In addition, PO can nominally be viewed as an electron–
acceptor, but the adjacent oxide can act as a π donor, such that the electronic effect of the
N–oxide is dependent on the extent of stability afforded by electron donation or
acceptance (Figure 4.1). However, the resonance effect of the N–oxide is greatest at the
2– and 4–position, and not at the 3–position. Hence, 4PNO can be expected to be more
stabilized than 3PNO due to position of nitrene. Moreover, for 4PNO, there are two
types of stabilization that can be envisioned to result from interaction between the nitrene
radical anion and the N–oxide, shown in Figure 4.1. In the π2σ state, the pyridinium can
serve as an electron pair acceptor, and the system can potentially be stabilized by
contribution from the quinone–like structure. Similarly, the σ2π state can be stabilized by
52
radical delocalization, creating a highly stable nitroxyl radical. Electron delocalization in
either state is such that it eliminates the formal charge separation of the n–oxides, which
could provide additional stabilization.
Figure 4.1. Resonance effect due to N-Oxide motif (Left) and the resonance structures of the π2α and α2π states of 4PNO (Right)
We have carried out a computational study of the electronic structures of 3PNO
and 4PNO, and an experimental investigation of their reactivity. Surprisingly, despite
the possible interactions between the nitrene radical anion and N–oxide in 4PNO, we do
not find significant differences in the computed electronic structure or reactivity of the
two isomers in the reaction with O2, CO2 and NO. However, a dramatic difference
between the ions is found in the reaction with CS2, as the 4–isomer undergoes reactivity
that is not observed for the 3–isomer, attributed to differences in accessibility of the oxide
end of the ion toward reaction. The results indicate that the resonance interaction
53
between the nitrene anion center and the n-oxide increases the nucleophilicity of the
oxygen in the n-oxide moiety.
4.2 Computational Detail
Structures and energies of the 2 and 2 states of 3PNO and 4PNO, NO, CS2
and possible reaction products were calculated at the B3LYP/6-31+G* level of theory.
Reaction energies correspond to differences in electronic energies between reactants and
products, and are not corrected for zero-point energies or thermal energy differences.
Calculations were carried out by using QChem,15 using the resources of the Center for
Computational Studies of Open-Shell and Electronically Excited Species
(iopenshell.usc.edu).
4.3 Results
The 4PNO- and 3PNO- radical anions are formed in high yields by dissociative
electron attachment to the corresponding azides (eq 4.2).
54
The azidopyridine-n-oxide precursors were prepared and supplied by our
collaborator James S. Poole from Ball State University. The synthesis and
characterization of these samples used in this work has been reported previously in
several occasions.6,16-19 The most significant impurity observed in these experiments
(with both isomers) is an ion with m/z 92, which is presumably the non-oxidized
pyridinylnitrene radical anion. The relative intensity of the m/z 92 ion is variable from
day to day, but decreases with time during the course of experiment run, indicating it
most likely results from ionization of a non-oxidized azidopyridine impurity.
Single point energies calculated at B3LYP /6–31+G* suggests that both isomers
have π2σ electronic ground states but 4PNO is more stable by 2.3 kcal/mol than 3PNO.
This electronic ground state is consistent to other aromatic nitrene radical anions like
benzoylnitrene radical anion. The σ2π states of 4PNO and 3PNO is 13.5 kcal/mol and
15.0 kcal/mol higher in energy than π2σ of 4PNO. The small difference in energy, 1.5
kcal/mol, between σ2π states of 4PNO and 3PNO reflects there is no significant
resonance effect, stabilization or destabilization, of the N–oxide in 4PNO as expected
(Figure 4.1).
4.3.1 Reactivity Studies
Reactions of 3PNO and 4PNO with O2, CO2, NO and CS2 are studied. When
reacted with O2 and CO2 both isomers produce a single peak at m/z 140 and at m/z 152
respectively. Upon CID, both of these product yield m/z 108 corresponding to the
molecular ion hence m/z 140 and m/z 152 are assigned to be O2 and CO2 adduct
55
respectively. Benzoylnitrene radical anion was found to be unreactive with CO2.13
Besides forming adducts and reacting differently with CO2 as compared to
benzoylnitrene, there is not much we could grasp on electronic structure from reactivity
of 3PNO and 4PNO with O2 and CO2.On the other hand, reactivity study of 3PNO
and 4PNO with NO and CS2 would help to interpret the electronic structure of
nitrenes.5,13
The results of reactivity studies of ions 3PNO and 4PNO with NO and CS2 are
shown in Table 4.1. Overall, there are no measurable differences in the rates of the
reactions for the two isomers. The efficiencies of the reactions with NO (approximately
0.2) are much higher than those found for reaction with CS2, but similar to that reported
previously for the reaction of NO with BzN (eff = 0.15).13 Although there are no
differences in the reaction rates for the two isomers with these reagents, there are very
large differences in the observed products. In the sections below, we consider the
differences in the products that are formed.
56
Table 4.1: Reaction Efficiencies and Product Branching Ratios for Reactions of 3PNO and 4PNO with NO and CS2
reagent result Ion assignment 3PNO– 4PNO–
NO Effa 0.21 0.20 m/z 138 [M+NO] – <1% 18% m/z 110 [M+NO–N2] – 44% 82% m/z 82 [M+NO–N2–CO] – 21% Nb
m/z 80 [M+NO–N2–NO] – 20% Nb
m/z 61 [M+NO–C3H3–N2]– 14% Nb
m/z 92 [M+NO–NO2] – Yc Yc
CS2 Effa 0.03 0.02 m/z 186 [M+CS2] – 83% 77% m/z 64 S2
– 15% 15% m/z 32 S– <1% <1% m/z 58 SCN– 2% 2% m/z 124 [M+CS2–CSO] – Nb 7% m/z 92 [M-O] – Nb Yc
aReaction efficiency, which corresponds to kexp/kADO bNot observed for this isomer. cProduct is observed in Q2, but the flow tube branching ratio cannot be determined due to the presence of impurities
4.3.1.1Reaction with NO
With nitric oxide, both 3PNO and 4PNO are observed to undergo N-O
exchange, similar to what is observed with PhN and BzN.13 Many additional products
are observed for the reaction of 3PNO with NO, but, as shown Table 1, they are
assigned to secondary fragmentation of the phenoxide anion. Possible product structures
are shown in eq 4.3. A notable difference in the reactivity of 3PNO and 4PNO with
57
NO is that reaction with 4PNO leads to significant amount of adduct ion, whereas only a
trace is observed with 3PNO. NO addition was not reported for either PhN or BzN,13
and is generally associated with reactions of closed-shell anions.5,20 Unfortunately, the
structures of the NO adducts are not known. However, charge distribution calculations
reported below find that there is more charge on the oxygen in 4PNO than in 3PNO,
which raises the possibility that the difference in the extent of adduct formation is due
4PNO forming an adduct at the oxygen, as opposed to at the nitrogen.
Both n-oxide anions are found to react with NO by oxygen atom transfer to form
the pyridinylnitrene radical anion, as shown for 4PNO in eq 4.4. Although the
branching ratios for these reactions in the flow tube cannot be determined due to the
presence of impurity ions, they were verified as products by using the reaction of NO
with mass-selected ions in Q2.
58
If the NO BDEs in 3PNO and 4PNO are similar to that in pyridine-n-oxide
(approximately 72 kcal/mol),21 then the oxygen atom transfer reaction would be
essentially thermoneutral. Alternatively, given that the reaction is carried out in Q2,
albeit under very low energy conditions, the oxygen atom transfer could be slightly
endothermic and translationally driven.
4.3.1.2 Reaction with CS2
Although the rates at which 3PNO and 4PNO react with CS2 are similar, there
are significant differences between the reaction products. In particular, some products
observed with 4PNO involve loss of the oxygen atom, and these products are not
observed with 3PNO. One product involving the oxygen is observed at m/z 124, which
is 16 Daltons higher than the reactant. The most likely assignment for this product is that
it arises from addition of sulfur and loss of oxygen, i.e. a sulfur-oxygen exchange. The
resulting products would be the n-sulfide and carbonyl sulfide, OCS.
The ion signal at m/z 92 is also likely due to reaction involving the oxygen. As in
the reaction with NO, we are unable to quantify the yield of the m/z 92 product due to
background presence of the pyridinylnitrene radical anion. However, considering 4PyN
is also observed to react with CS2, it appears that m/z 92 is, in fact, formed in relatively
59
high yield (comparable to the yield of S2). This is also indicated by the results on “clean”
days, where the mass spectrum includes minimal amounts of the impurity. The formation
of m/z 92 in the reaction of 4PNO with CS2 was also confirmed by using mass-selected
ions in Q2.
The identity of the m/z 92 is not known unequivocally. Although the ion with m/z
92 could be the pyridinylnitrene radical anion, there is a second possibility. The ion
CS2O is isobaric with the pyridinylnitrene radical anion, and could, in principle be
formed by oxygen-anion transfer with 4PNO, as shown in eq 4.5.
Our experimental results suggest both structures are present. Evidence for the
formation of CS2O comes from the isotopic distribution of the product. Figure 4.2
shows a mass spectrum of 4PNO taken on an exceptionally clean day (with minimal m/z
92 contamination), with and without the addition of CS2. The background signal of m/z
92 in the spectrum is less than 10 kcps. However, upon addition of CS2, the signal
increases to nearly 100 kcps. Most importantly, the signal at m/z 94 also increases, to
nearly 8% of the m/z 92 signal, which agrees well with the value of 9% expected for
CS2O. Therefore, the M+2 isotope peak indicates that there is sulfur present in the m/z
92 ion.
60
However, reactivity evidence suggests the presence of 4PyN as well. Figure 4.3
shows the mass spectra for reaction of m/z 92 ion, formed by reaction of 4PNO with CS-
2, with NO. The formation of m/z 94 is clear evidence for the presence of a nitrene
radical anion, 4PyN.
Figure 4.2. The m/z 92 region of the mass spectra of 4PNO before (dashed) and after (solid) addition of CS2
61
Figure 4.3. The m/z 92 region of the mass spectra of 4PNO reaction with CS2 (dashed) and after addition of NO (solid)
Computationally, both reactions are computed to energetically favorable. At the
B3LYP/6-31+G* level of theory, the formation of CS2O and the triplet pyridinylnitrene
(eq 4.5) is computed to be exothermic by 72 kcal/mol, or more than 3 eV! For oxygen
atom transfer, there are multiple thermochemically accessible pathways. Direct transfer
to form CS2O (eq 4.6a) is computed to be exothermic by 7.7 kcal/mol. A second
pathway, shown in eq 4.6b, involves the formation of CO + triplet S2, and is computed to
be exothermic by 9.9 kcal/mol.
62
Deoxygenation of teritiary-amine-n-oxides in solution22-24 has been proposed to
occur by a mechanism such as that shown in Scheme 4.3, leading to the formation of the
dithiiranone as in eq 4.6a. In solution, the dithiiranone can be hydrolyzed to CO2 +
HSSH22 or can be utilized as a sulfur transfer reagent25 .
In the gas phase, the reaction of oxygen atom with CS2 gives CS + SO as the
major products.26 However, CO has also been observed in the reaction, 27 indicating that
CO + S2 is a possible decomposition pathway of CS2O. In fact, CO + S2 is energetically
Scheme 4.3
63
the most favored pathway26 but is slow because there is a slight (6.6 kcal/mol) barrier26
for the initial formation of CS2O. However, this barrier can be overcome in the reaction
of CS2 with 4PNO by the energy released upon formation of the initial ion/molecule
complex in combination with the oxygen atom transfer exothermicity. Therefore, oxygen
atom transfer to form CO and S2 products should be able to occur for 4PNO. Formation
of CS + SO is approximately 3 eV less favorable26 than formation of CO + S2 and is
therefore highly endothermic in the reaction of 4PNO with CS2.
As noted above, sulfur-oxygen exchange is also observed in the reaction of CS2
with 4PNO. The mechanism of sulfur-oxygen exchange likely involves a first step
similar to that for oxygen atom transfer, addition of the oxygen to the center carbon of
CS2, as shown in Scheme 4.4. However, instead of forming the disulfide bond as in
dithiiranone formation, a N-S bond is formed.
A small amount of NCS product is observed in the reaction of CS2 with both
3PNO and 4PNO. NCS has been observed previously in reactions of CS2 with closed-
shell, nitrogen-based anions,5,28,29but has not been reported previously for reactions of
open-shell anions. The mechanism is presumably similar to that shown in Scheme 4.4,
although occurring at the nitrogen. The other products observed with CS2 (S, S2 and
CS2 adduct) are similar to those observed previously with nitrene radical anions.13
64
4.3.1.3 Comparison of Isomers: Oxygen Nucleophilicity
There are significant differences in the reactions that occur between ions 3PNO
and 4PNO and CS2. Specifically, 4PNO undergoes sulfur-oxygen exchange and
oxygen-atom and/or oxygen-anion transfer reactions that are not observed with 3PNO.
The common feature of these reactions is that they all involve initial nucleophilic attack
of the oxygen in the anion at the carbon of the carbon disulfide. The fact that 4PNO
undergoes reaction at the oxygen whereas 3PNO does not reflects important differences
in their electronic structures.
The best example that illustrates the electronic structure differences between the
ions is the oxygen-transfer reaction with CS2. Although carbon disulfide is known to
deoxygenate tertiary-amine-n-oxides,22 the reaction is generally not observed with
aromatic-n-oxides. Therefore, the lack of oxygen transfer with 3PNO is consistent with
the expectation that the substituent in the meta position does not interact with the n-oxide
moiety. Similarly, the nitrene anion para to the n-oxide can serve as a -donor, as shown
Scheme 4.4
65
in Figure 4.1, which makes the oxygen in the 2 state more nucleophilic. Hammett
analysis of the deoxygenation of aniline-n-oxides in solution has shown22 that the
reaction is favored by strong electron donating substituents, which increase the
nucleophilicity of the oxygen. Apparently, the nitrene radical anion is such a strong -
electron donor that it even enables oxygen transfer in the normally inert aromatic-n-
oxides.
The difference in the reactivity cannot be attributed to differences in overall
thermochemistry for the reactions. All of the reactions observed for 4PNO are also
computed to be exothermic for 3PNO, although they don’t occur. For example, the
sulfur-oxygen exchange reaction observed for 4PNO (Scheme 4.4) is computed to be
exothermic by 45 kcal/mol. Similarly, the sulfur-oxygen exchange reaction for 3PNO is
computed to be exothermic by 37 kcal/mol, but does not occur at all. Therefore, the
difference in reactivity is more likely due to kinetic differences that result from
differences in electronic structure.
The increased nucleophilicity of the oxygen in 4PNO apparent in the reactivity
studies is consistent with the computed charge distributions in the 2 states. The
B3LYP/6-31+G* computed Mullikin charges at the heavy atoms in 3PNO and 4PNO
are shown in Figure 4.4.
66
Figure 4.4. Calculated Mullikin charge distributions in 3PNO, 4PNO and pyridine-n-oxide. Charges on hydrogen have been summed into the heavy atoms.
Although both ions have increased charge density at the oxygen than is found in
pyridine-n-oxide there are some differences in charge densities at the carbon atoms, the
most significant difference is for the oxygen, where the calculated charge is larger in in
4PNO than in 3PNO, which accounts for the increased reactivity at that site. Increased
reactivity at the oxygen accounts for the observation of either oxygen atom (eq 4.6) or
oxygen ion (eq 4.5) transfer with
As suggested above, the difference in the charge density at the oxygen may also
account for the difference in the extent of adduct formation with nitric oxide. This is
most likely the case if the adduct is an electrostatic complex. However, as noted, the
structure of the adduct is not known. Aside from the extent of adduct formation, there is
little difference in the reactivity of with nitric oxide. The differences in the observed
products can be attributed to differences in the ability of the resulting phenoxide ion to
fragment.
67
4.4 Conclusion
The reactivity of 3PNO and 4PNO, particularly with CS2, show that there are
significant differences in their electronic structures, which can be understood as resulting
from the resonance interaction between the nitrene anion and the n-oxide moiety in
4PNO, and the lack thereof in 3PNO. In the 2 state, the monovalent nitrogen anion
is a strong electron donor, which increases the charge density on the oxygen, as shown
in Figure 4.2. The result is consistent with the conclusions based on condensed-phase
studies that oxygen atom transfer is favored by -donors.22 However, the reaction has not
been observed previously for aromatic n-oxides. This work shows that oxygen atom
transfer for aromatic n-oxides can occur with sufficiently strong donors. The
differences in the electronic structures of 3PNO and 4PNO do not result in differences
in reactivity with NO, although there are differences in the stabilities of the resulting
products.
68
4.5 References
(1) Platz, M. Acc. Chem. Res. 1995, 28, 487.
(2) Horner, L.; Christma.A . Angew. Chem. Int. Ed. 1963, 75, 707.
(3) Belloli, R. J. Chem. Educ. 1971, 48, 422.
(4) Borden, W.; Gritsan, N.; Hadad, C.; Karney, W.; Kemnitz, C.; Platz, M. Acc. Chem. Res. 2000, 33, 765.
(5) Rau, N.; Welles, E.; Wenthold, P. Journal of the American Chemical Society 2013, 135, 683.
(6) Hostetler, K.; Crabtree, K.; Poole, J. J. Org. Chem. 2006, 71, 9023.
(7) Drzaic, P.; Brauman, J. J. Am. Chem. Soc. 1984, 106, 3443.
(8) Drzaic, P.; Brauman, J. J. Phys. Chem. 1984, 88, 5285.
(9) McDonald, R.; Davidson, S. J. Am. Chem. Soc. 1993, 115, 10857.
(10) Travers, M.; Cowles, D.; Clifford, E.; Ellison, G. J. Am. Chem. Soc. 1992, 114, 8699.
(11) Wijeratne, N.; Da Fonte, M.; Ronenius, A.; Wyss, P.; Tahmassebi, D.; Wenthold, P. J. Phys. Chem. A 2009, 113, 9467.
(12) Wijeratne, N.; Wenthold, P. J. Am. Soc. Mass. Spectrom. 2007, 18, 2014.
(13) Wijeratne, N.; Wenthold, P. J. Org. Chem. 2007, 72, 9518.
(14) Wijeratne, N.; Wenthold, P. J. Phys. Chem. A 2007, 111, 10712.
(15) Shao, Y.; Molnar, L.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.; Brown, S.; Gilbert, A.; Slipchenko, L.; Levchenko, S.; O'Neill, D.; DiStasio, R.; Lochan, R.; Wang, T.; Beran, G.; Besley, N.; Herbert, J.; Lin, C.; Van Voorhis, T.; Chien, S.; Sodt, A.; Steele, R.; Rassolov, V.; Maslen, P.; Korambath, P.; Adamson, R.; Austin, B.; Baker, J.; Byrd, E.; Dachsel, H.; Doerksen, R.; Dreuw, A.; Dunietz, B.; Dutoi, A.; Furlani, T.; Gwaltney, S.; Heyden, A.; Hirata, S.; Hsu, C.; Kedziora, G.; Khalliulin, R.; Klunzinger, P.; Lee, A.; Lee, M.; Liang, W.; Lotan, I.; Nair, N.; Peters, B.; Proynov, E.; Pieniazek, P.; Rhee, Y.; Ritchie, J.; Rosta, E.; Sherrill, C.; Simmonett, A.; Subotnik, J.; Woodcock, H.; Zhang, W.; Bell, A.; Chakraborty, A.; Chipman, D.; Keil, F.; Warshel, A.; Hehre, W.; Schaefer, H.; Kong, J.; Krylov, A.; Gill, P.; Head-Gordon, M. Phys Chem Chem Phys. 2006, 8, 3172.
(16) Itai, T.; Okusa, G.; Sako, S. Chem. Pharm. Bull. 1963, 11, 1146.
69
(17) Itai, T.; Natsume, S. Chem. Pharm. Bull. 1964, 12, 228.
(18) Crabtree, K.; Hostetler, K.; Munsch, T.; Neuhaus, P.; Lahti, P.; Sander, W.; Poole, J. J. Org. Chem. 2008, 73, 3441.
(19) Sawanishi, H.; Tajima, K.; Tsuchiya, T. Chem. Pharm. Bull. 1987, 35, 4101.
(20) Chacko, S.; Wenthold, P. Mass Spectrom. Rev. 2006, 25, 112.
(21) Afeefy, H. Y.; Liebman, J. F.; Stein, S. E.; National Institute of Standards and Technology, Gaithersburg, MD 20899: retrived February 11, 2014.
(22) Yoshimura, T.; Asada, K.; Oae, S. Bull. Chem. Soc. Jpn. 1982, 55, 3000.
(23) Hamana, M.; Nakashima, S.; Umezawa, B. Chem. Pharm. Bull. 1962, 10, 969.
(24) Nakayama, J.; Ishii, A. Adv. Heterocycl. Chem. 2000, 77, 221.
(25) Zipplies, M.; Devos, M.; Bruice, T. J. Org. Chem. 1985, 50, 3228.
(26) Saheb, V. J. Phys. Chem. A 2011, 115, 4263.
(27) Hudgens, J.; Gleaves, J.; McDonald, J. J. Chem. Phys. 1976, 64, 2528.
(28) Barlow, S.; Bierbaum, V. J. Chem. Phys. 1990, 92, 3442.
(29) Depuy, C. Org. Mass Spectrom. 1985, 20, 556.
70
CHAPTER FIVE: MASS SPECTROMETRIC STUDY OF THE DECOMPOSITION PATHWAYS OF CANONICAL AMINO ACIDS AND α-LACTONES IN THE GAS
PHASE
5.1 Introduction
The significance of amino acids to the world around us, and to life itself, cannot
be overstated, and, therefore, it is not surprising that their structures, properties, and
reactivity are exceptionally well-characterized, at least in solution. However, owing to
their general lack of volatility, much less is known about the structure and reactivity of
amino acids in the gaseous state. Gaseous amino acids are rare, but would be highly
significant if reports of interstellar glycine, 1,2 for example, can be confirmed. In a more
practical sense, the gas phase serves as a reasonable approximation for a low dielectric,
hydrophobic environment, and so investigation of the gas-phase (solvent free) properties
of amino acids provides insight into the structures of amino acids in lipid membranes.3
Finally, regardless of any biological significance, results of studies of gaseous amino
acids can be compared to those for substituted carboxylic acids to determine, inter alia,
the effect of the amino group on the gas-phase structure and reactivity.
One of the most important differences between gaseous and condensed-phase
amino acids is that, because charge separation is unfavorable in the gas phase, the
solvent-free molecules will not have zwitterionic structures, Z, like those in condensed
phase, but will have non-ionic, canonical (C) structures instead.4-7 In fact, zwitterionic
71
structures of gas-phase amino acids are so unexpected that many mass spectrometric
studies have been carried out to try to elucidate those factors, such as solvation or ion-
pairing, that can lead to stable ionic structures in various amino acids.8
Recent advances in spectroscopic methods in the last decade have provided more
detailed insight into the gas-phase structures, including details of the modes of internal
hydrogen bonding.9-11 The challenges in these experiments are two-fold: on one hand,
because the spectroscopy experiments generally require a good chromophore for
ultraviolet absorption, they work best for amino acids with aromatic side chains.
However, those amino acids are among the less volatile, and therefore creating gaseous
samples is difficult. Smaller, aliphatic amino acids are more volatile, but lack a good
chromophore to facilitate resononace-2-photon absorption, which is generally required
for these types of experiments. It is a testament to modern spectroscopic technology that
we do have spectra for both aromatic and aliphatic amino acids.12-15 Gas-phase
photoionization of aliphatic amino acids has also been reported.16
Amino acids have been extensively studied using mass spectrometry. The
development of ionization techniques like electrospray ionization (ESI), matrix assisted
laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) has
made the desorption and ionization of non–volatile substrate, like AA, possible allowing
characterization using mass spectrometry (MS). However, in these studies, the amino
72
acids are typically ionized in some way, such as via protonation or metallation to create
positively-charged ions, or by deprotonation to form negatively-charged ions.
Consequently, the properties of ionized amino acids investigated by using mass
spectrometry are generally not comparable to those of the “neutral” (zwitterionic or
canonical) amino acids. Here we describe an investigation of canonical amino acids by
using “charge-remote fragmentation,” 17,18 where dissociation of an ion occurs, as the
name suggests, at locations remote from the charge site. Charge-remote fragmentation is
commonly used as an analytical tool to determine the structures of biological molecules.
Lipids17,18 are particularly amenable to this technique, as they fragment in the
hydrocarbon chains. However, charge-remote fragmentation has also been used to
examine structures of other biologically relevant molecules such as drugs and their
metabolites and peptides.17,18
Adams and Gross19 have proposed that charge-remote fragmentation, where the
charge does not participate in the fragmentation, is analogous to thermolysis of the gas-
phase neutral molecule. If this is true, then it may be possible to use charge-remote
fragmentation electrosprayed ions as an alternative to pyrolysis in order to study the
gaseous decomposition of otherwise non-volatile organic molecules. In this study, we
examined sulfonated phenylalanine, PheSO3, a sulfonate anion with a separate canonical
amino acid moiety.
73
The gas-phase reactivity of amino acids is similarly poorly known. The lack of
volatility of amino acids has made even nominally simple experiments such as gas-phase
pyrolysis exceedingly difficult, and these experiments generally require generation of the
amino acid in situ by pyrolysis of appropriate volatile precursors. For example, Chuchani
and co-workers20 reported that they could examine the pyrolytic dissociation of amino
acids generated by decomposition of the corresponding ethyl ester derivatives (eq 5.1).
By using this approach, they found that N-substituted glycines, such as N,N-dimethyl-,20
N-phenyl-21 and N-benzylglycines,22 for example, dissociate by loss of CO2, as shown in
eq 5.1, in the gas phase. In contrast, Al-Awadi and co-workers found that gaseous N-
phenylalanine does not undergo decarboxylation upon pyrolysis, but dissociates into
aniline, CO, and acetaldehyde, as shown in eq 5.2,23 in a process that is similar to what is
commonly observed for α-substituted carboxylic acids (eq 5.3).24-36
74
By using mass spectrometry, we have examined the decomposition of the gaseous
amino acid, PheSO3, which contains a sulfonate charge and a canononical -amino acid.
We show that PheSO3 dissociates initially by fragmentation of the amino acid by the
pathway shown in eq 5.3 to form the α-lactone, and direct decarboxylation (eq 5.1) is not
observed. The α-lactone product is found to fragment by decarbonylation, as expected
(eq 5.3) but is also found to undergo decarboxylation to form the styrene product.
Electronic structure calculations find that the presence of the sulfonate charge has little
effect on the energetics of these dissociation pathways, indicating that the results can be
fairly interpreted as representative of what would happen with the non-ionized derivative.
5.2 Experimental
Experiments were carried out using a commercial LCQ-DECA (Thermo Electron
Corporation, San Jose, CA) quadrupole ion trap mass spectrometer, equipped with
electrospray ionization (ESI). Ions were introduced by spraying dilute aqueous solutions
of commercially available substrates, except for the cis-cinnamic acid and 4-
(acryloyloxy)benzenesulfonate, which were synthesized as described below; solution
details are provided as Supporting Information. Electrospray and ion focusing conditions
were varied so to maximize the signal of the ion of interest. Dissociation of ions can be
carried out during ion injection by using high injection voltages, or can be carried out by
using MSn experiments with mass-selected ions in the cell, with the helium buffer serving
as the collision target. Reactant ions for CID were isolated at qz = 0.250 and with a mass-
width sufficient to avoid off-resonance excitation of the mass-selected ions. The energy
75
of collision-induced dissociation (CID) in the cell is reflected in the “normalized collision
energy,” which ranges from 0 – 100%.
5.3 Synthesis
5.3.1 (Z)-4-(3-ethoxy-3-oxoprop-1-en-1-yl)benzenesulfonate (deprotonated ethyl 4-sulfo-cis-cinnamate)
A solution of ethyl diphenyl phosphoacetate (0.16 g, 0.50 mmol) in THF (30 ml)
was treated with Triton B (0.27 ml, 0.60 mmol) at -78 °C for 15 min. A solution of p-
sulfonated benzaldehyde (0.12 g, 0.60 mmol in 5 ml MeOH) was then added dropwise,
and the resulting mixture was stirred at -78 °C for 1 hr. The reaction was quenched with 5
ml of D.I water. ESI mass spectra of the crude product indicated a mixture of m/z 255
(the sulfonated ethyl cinnamate) and m/z 249, which is assigned to a (PhO)2PO2 by-
product. NMR of the crude mixture in D2O contained doublets at 6.0 and 6.98 ppm (J =
12.4 Hz), which could be assigned to the cis-cinnamate based on comparison with
previously reported cis-structures. In contrast, the clearly observed NMR peak for the
authentic trans isomer is a doublet at 6.49 ppm with J = 15.4. From the NMR spectrum
of the cis-sample, we estimate a cis/trans ratio of about 98:2, consistent with the
selectivities obtained previously using this procedure. The ethyl ester was introduced
into the mass spectrometer by electrospray and converted to the carboxylic acid by CID,
and elimination of ethylene.
76
5.3.2 4-(acryloyloxy)benzenesulfonate (PASO3)
4-(acryloyloxy)benzenesulfonate was prepared by mixing 1 mL of 4-
hydroxybenzenesulfonic acid sodium salt dihydrate with 3 mL of acryloyl chloride
(CH2=CHCOCl). The neat mixture was stirred for about 15 minutes at 35º, and 1 mL of
(i-Pr)2Net was added. The solution was stirred approximately 15 minutes, after which
one drop of the crude mixture was added to 1 mL of a 50/50 mixture of water and
methanol and examined by ESI mass spec. The main products observed in the mass
spectrum of the crude mixture are the hydroxybenzenesulfonate, a product with m/z 253,
which is likely HO3SC6H4SO4, and a product at m/z 227, which is the phenyl acrylate.
5.4 Computational Methods
Amino acid conformations were surveyed by using the Monte Carlo Multiple
Minimum search method, with the MMFFs force field, as implemented in MacroModel
(version 9.6).37 Multiple starting conformations were utilized, including canonical and
zwitterionic structures, to improve the chances of finding the key low energy structures.
All of the unique conformations calculated with the MMFFs force field to be within 100
kJ/mol of the lowest energy structure were identified and used as starting points for
B3LYP/6-31+G* geometry optimizations.
Stationary points were confirmed to be minima or saddle points from the
computed vibrational frequencies. Reported energies are electronic energies, computed
at the MP2/6-311+G** level of theory at the B3LYP geometries, designated MP2/6-
311+G**//B3LYP/6-31+G*, unless noted, and are not corrected for zero-point energies
or thermal energy contributions. The larger basis set is used to provide a good
77
description of the van der Waals interactions and polarization of charge. Calculations
were carried out using QCHEM version 4.038 and Gaussian.39
5.5 Results
The mass spectrum of an aqueous solution of PheSO3, shown in Figure 5.1a, is
very clean, with essentially only peaks corresponding to PheSO3. Considering the large
difference in a acidities of sulfonic and carboxylic acids,40 it is not surprising that the
sulfonate structure, depicted as PheSO3, is computed (B3LYP/6-31+G*) to be more
than 20 kcal/mol more stable than the carboxylate structure, similar to what has been
reported previously for sulfated peptides.41 Although mixtures of ion isomers have been
found for amino acid anions when the acidities of the different groups are similar,42,43 the
large difference in the acidities of the carboxylic and sulfonic acids should result in an
ion that consists of a sulfonate charge with separate amino acid moiety. Although the
extent of interaction between the charge and the reactive group is always a question in
these studies, the distance to the charge is too great for any significant interaction to
occur. In addition, the saturated carbons that separate the amino acid from the ring
prevent any conjugative interactions with the charge.
78
Figure 5.1. a) ESI and b) CID mass spectra of PheSO3. The CID for spectrum was
carried out with a normalized collision energy of 25%.
Calculations have been carried out to determine the structure of PheSO3
in the
gas phase. A conformational search identified more than 20 unique low-energy
conformations (within 50 kJ/mol of minimum) of the ion, and all but one which have
canonical structures. Qualitative depictions of the 5 lowest energy canonical
conformations and the lowest energy zwitterionic structure are shown in Figure 5.2.
Figure 5.2 also shows the relative energies of each stationary point, optimized at the
B3LYP/6-31+G* level of theory. The most stable conformation has the carboxylic acid
moiety anti to the aromatic ring, with a hydrogen bond between the –OH proton and the
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
150 170 190 210 230 250 270 290
M
M-NH3
M-NH3-COM-NH3
-CO2
a
b
79
amine nitrogen. Indeed, the anti-conformation, when possible, is generally preferred for
the structures examined in this work. The conformation with the –COOH and aromatic
ring gauche is higher in energy by about 2.5 kcal/mol. Alternate hydrogen bonding
arrangements, such as NH- or OH to carbonyl oxygen, or NH to hydroxyl hydrogen, are
higher in energy by more than 4 kcal/mol. The most important conclusion from this
computational survey is that the lowest energy zwitterionic structures are significantly
higher in energy than the lowest canonical forms. Figure 5.2 also shows that the relative
energies are similar whether using either DFT or ab initio methods.
Figure 5.2. Low-energy conformations of the amino acid moiety of PheSO3 and their
relative energies, in kcal/mol, computed at the B3LYP/6-31+G* and MP2/6-311+G** (in parentheses) level of theory.
5.5.1 Dissociation of PheSO3
The CID spectrum of PheSO3 is shown in Figure 5.1b. The major fragments
observed are readily assigned as resulting from dissociation of the canonical amino acid
backbone. In particular, the main dissociation processes all involve loss of NH3 to form
an ion with m/z 227, which subsequently fragments by loss of either CO, to form m/z
199, or CO2, to form m/z 183. That the m/z 183 and 199 ions come from the M-NH3
80
anion is confirmed by two additional results. First, although m/z 183 and 199 are major
products observed at the energy used for CID in Figure 5.1b, the M-NH3 ion is the only
product observed at very low energies, consistent with a sequential dissociation. Second,
CID of the isolated M-NH3 anion, generated either by CID during the ESI injection stage,
or by CID in the course of an MS/MS/MS experiment, verifies that it dissociates by loss
of CO and CO2 in a nearly 1:1 ratio as observed in Figure 5.1b. Therefore, the loss of
NH3 is the only pathway that can be uniquely assigned to PheSO3. Direct loss of CO2,
which has been found previously for pyrolysis of gas-phase amino acids (eq 5. 1),20 is not
observed upon activation of PheSO3.
Although the structure of the M-NH3 product is not known, the most likely
products to consider are those that are stable products formed by minimal rearrangement.
Therefore, the most likely products are either a cis- or trans-cinnamic acid (CASO3), an
α-lactone (αLSO3) or the phenyl acrylate (PASO3
). Other products are possible but
would require more extensive rearrangement.
CID of deprotonated phenyl alanine is known to result in formation of cinnamic
acid.44-46 Similarly, dissociation of PheSO3 by elimination across the ethylene backbone
would result in the formation of a cinnamic acid, CASO3, as shown in eq 5. 4. However,
multiple lines of evidence argue against the cinnamic acid. First, ammonia loss from d3-
labeled PheSO3, formed by H/D exchange using a mixture of D2O/CD3CO2D as the
solvent, occurs by loss of ND3 only, and loss of ND2H, NDH2 or NH3 is not observed to
any detectable extent at any collision energy.
81
This rules out the possibility rules out elimination across the backbone as shown
in eq 5.4. It is also evidence against other, more complicated rearrangement processes
involving non-amino acid portions of the molecule. However, cinnamic acid formation
from PheSO3 does not require dissociation across the backbone. An alternate
mechanism, as shown in eq 5.5, proceeds via a zwitterionic intermediate.
82
Therefore, we have examined the CID behavior of authentic trans- and cis-
cinnamic acids to compare with the product obtained from PheSO3. For this
experiment, sulfonated trans-cinnamic acid was formed by ESI of an aqueous solution of
the commercially available sulfonyl chloride, and the cis-cinnamic acid was formed in
situ by CID of the corresponding ethyl ester. A comparison of the CID spectra of the m/z
227 product obtained from PheSO3 and the authentic cinnamic acids, measured with a
normalized collision energy of 34%, is shown in Figure 5.3. Whereas all ions lose CO2
as the major fragmentation pathway, significant differences are evident. For example, the
CASO3 ions do not lose CO to any appreciable extent, and lose only SO2 (to form m/z
163) in addition to losing CO2. Moreover, although the m/z 183 ion that corresponds to
PheSO3-NH3-CO2 is found to lose SO2 upon CID (vide infra), the CASO3
ions
undergo facile loss of CO2 and SO2 to form m/z 119 in much higher yield than is found
for the M-NH3 ion. Finally, although the authentic CASO3 ions dissociate by loss of
CO2, they do so only at energies much higher than those needed for dissociation of
PheSO3. The amounts of the m/z 163 and m/z 119 products observed in Figures 5.3a,
5.3b and 5.3c indicate that CASO3 constitutes at most about 5% of the M-NH3 product.
83
Figure 5.3. CID spectra of m/z 227 ions obtained a) by CID of PheSO3, and from
sulfonated b) cis and c) trans cinnamic acid
As shown in eq 5.6, the zwitterionic intermediate could also rearrange through
nucleophilic attack at the ipso-position followed by a Grob-like fragmentation to give
PASO3.
84
NH3
O
O
+
_
SO3_
SO3_
O
+ NH3 (5.6)
O
PASO3_
Although PASO3 is likely a relatively stable product, it can be ruled out for
multiple reasons. First, it is inconsistent with the CID results, in that it cannot readily
lose either CO or CO2. Second, formation of the phenyl acrylate would require a high-
energy, non-aromatic transition state (vide infra). Ultimately, the formation of PASO3
was ruled out by using an authentic sample. The primary dissociation pathway of
PASO3 is loss of the acryloyl group (CH2=CHCO) to form the phenoxyl radical, loss of
CO or CO2 does not occur at all. In contrast, formation of the α-lactone is consistent with
all of the data. Formation of the lactone, either by a concerted loss of NH2H or through
a zwitterionic intermediate (eq 5.7) involves loss of only the exchangeable protons, and is
therefore consistent with the results for the deuterated ions.
85
Loss of CO like that observed here is a well-known decomposition pathway for α-
lactones,24-36 and therefore loss of CO from the M-NH3 anion is consistent with the
presence of the α-lactone. Although decarboxylation is generally not considered a
decomposition pathway for that type of structure, it is not unprecedented,47 and
computational studies described herein predict that it is expected to occur for αLSO3.
Based on these results, we conclude that the M-NH3 ion found upon CID of
PheSO3 is most likely the α-lactone, αLSO3
. Although α-lactones are nominally high-
energy reactive species, they have previously been proposed as products in the CID of -
substituted carboxylates in mass spectrometry experiments,48-51 which is essentially how
it is shown being formed via the zwitterion in eq 5.7. The difference between the
previous studies and this work is that the α-lactone formed upon charge remote
fragmentation of PheSO3 is ionic, and therefore detectable by mass spectrometry.
86
Previous studies of halogenated carboxylates 48-51 have inferred α-lactone structures
based on the observation of halide products.
5.5.2 Dissociation of the α-Lactone, αLSO3
As described in the section above, the PheSO3 - NH3 product, assigned to be the
α-lactone (αLSO3), dissociates by loss of either CO or CO2. Loss of CO from α-
lactones is well-known and predicted to occur with a barrier of about 28-30 kcal/mol52 to
form the corresponding aldehyde or ketone (eq 5.3). The CO-loss product obtained upon
CID of αLSO3 in this work is found to undergo further dissociation by loss of 29 mass
units, which we assign to HCO as shown in eq 5.8.
Dissociation to form the benzyl radical product, BzSO3, is also observed upon
CID of control substrates, such as sulfonated phenylpropionic acid or phenethylamine (eq
5. 9), and so it is not surprising that it is also observed for the aldehyde.
87
The loss of CO2 has not been reported previously for the gas-phase decomposition of α-
lactones, although it has been found to occur, along with decarbonylation, upon
photolysis of the bis-n-butyl-substituted α-lactone in solution,47 forming the olefin (eq
5.10).
By comparing CID spectra of the m/z 183 product with an authentic sample, we
have confirmed that the product formed upon decarboxylation of αLSO3 is similarly the
corresponding styrene (eq 5.11).
88
In summary, we have found that the dissociation of the gas-phase amino acid,
PheSO3, results in formation of an α-lactone, αLSO3
, which dissociates by
decarbonylation to form the aldehyde, and by decarboxylation accompanied by hydrogen
shift to form the styrene.
5.6 Discussion
The results described in the previous section are the first studies of the
dissociation of a simple (non-N-substituted), canonical amino acid in the gas phase. The
dissociation pathway for the amino acid is similar to that observed previously for
substituted alanine23 and other substituted carboxylic acids,24-36 but is very different from
what has been reported for glycine derivatives.20-22 The difference in dissociation
pathways between glycine and alanine derivatives has been noted previously22 but has not
been explained satisfactorily.
That the decomposition of PheSO3 involves only the amino acid moiety and not
the SO3 group is evidence that the sulfonate charge does not participate in the
dissociation processes. Fragmentation of the SO3 group is not even observed for any of
the dissociation products, except for the sulfonated styrene shown in eq 5.11, which
dissociates by loss of SO2. Therefore, the experimental results indicate that the sulfonate
group is serving extensively as a charge carrier, and does not significantly affect the
possible dissociation pathways. Although the dissociation pathway, loss of NH3, is the
same as what is found for deprotonated phenylalanine,44-46 the product formed with that
ion is deprotonated cinnamic acid. Control experiments in this work show that cinnamic
acid is not formed upon dissociation of PheSO3. The mechanism proposed for
89
deprotonated phenylalanine44-46 involves intramolecular proton transfer to the carboxylate
and subsequent proton transfer to the amine, and therefore the charge center is
extensively involved in the process. The lack of formation of cinnamic acid with
PheSO3 suggests the charge center is not participating in the dissociation process.
Deprotonated tyrosine also undergoes CID by loss of ammonia,53 possibly via the
phenoxide ion.
Electronic structure calculations predict that the sulfonate charge does not have a
large effect on the energies of the reaction pathways for PheSO3. A comparison of the
relative energies of observed and other possible products in the dissociation of PheSO3
and neutral phenylalanine, Phe, calculated at the MP2/6-311+G**//B3LYP/6-31+G*
level of theory, are shown in Table 5.1. Table 5.1 shows that the relative energies for all
of the possible dissociation products for PheSO3 and Phe are very similar, with
differences for the primary process of less than 3 kcal/mol.
Table 5.1. Computed Reaction Energies for Dissociation Pathways of PheSO3 and Phea
Relative Energies Products PheSO3
Phe Amino acid (reactant) 0.0 0.0 ArCH2C(CO)CH + NH3 (α-lactone, eq 5. 7) 47.0 44.6 ArCH=CHCO2H + NH3 (trans cinnamic acid, eq 5. 4)
17.7 16.4
ArCH2CH2NH2 + CO2 1.1 -4.6 ArCH2CHO + CO + NH3 34.9 31.9 ArCH=CH2 + CO2 + NH3 15.9 11.4 ArOC(O)CH=CH2 + NH2 39.4 39.0
aElectronic energy differences between products and the amino acid reactants, computed at the MP2/6-311+G**//B3LYP/6-31+G* level of theory.
90
5.6.1 Amino Acid Dissociation
A potential energy surface for the dissociation of PheSO3, shown in Figure 5.4,
provides insight into the product selectivity. Because CID is a kinetic process, the most
important features of the potential energy surface are the activation barriers. Therefore,
we have calculated the barriers for NH3 and CO2 loss from PheSO3 and Phe at the
MP2/6-311+G**//B3LYP/6-31+G* level of theory. Despite multiple attempts, we could
not find any transition states that directly connect the canonical structure of the amino
acid with the expected deamination or decarboxylation products. However, insight into
the mechanism was obtained from multidimensional potential energy surface
calculations. We find that loss of NH3 or CO2 occurs effectively by dissociation of the
zwitterionic structure, in that proton transfer from the carboxylic acid to the amino-group
occurs very early in the process at energies well below those required for dissociation.
91
Figure 5.4. Calculated energetics for the dissociation of PheSO3
(X = SO3) and Phe (X
= H). Values correspond to electronic energies and geometries calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of theory a) not a stable geometry; corresponds to an
amino acid geometry the same as that for zwitterionic PheSO3; b) computed utilizing a 3
kcal/mol barrier for the reverse reaction, obtained from a relaxed surface scan; see Supporting Information.
Loss of NH3 from Phe to form the α-lactone is estimated to have a barrier of about
3 kcal/mol higher than the reaction energy, about 46 kcal/mol. The mechanism for
ammonia elimination involves an intramolecular SN2 reaction (eq 5.12), where the
carboxylate displaces the amine leaving group in the zwitterionic structure. Therefore,
the dissociation of PheSO3 to form the α-lactone is analogous to the elimination
reactions of α-halocarboxylates that have been reported previously.50,54-60 In an ab initio
92
study, Davidson and co-workers61 found that there is no barrier in excess of the
dissociation energy for formation of the α-lactone from chloroacetate, whereas a small
barrier was calculated for the zwitterionic structure of Phe in this work.
The barrier for elimination of NH3 to form the cinnamic acid, not shown on Figure 5.4, is
calculated to be 68 kcal/mol, which accounts for why that process does not occur.
However, given that charge remote fragmentation is analogous to pyrolysis, that
cinnamic acid is not formed is not surprising considering that pyrolysis of alkylated
amines does not occur by concerted elimination of NH3, but results in the formation of
imines.62 Similarly, we have calculated the barrier for formation of the phenyl acrylate,
PASO3, as shown in eq 5. 6. Whereas for the neutral phenyl alanine we find a concerted
transition state for rearrangement accompanied by ammonia loss, the transition state for
the sulfonated version has the ammonia completely dissociated, and is part of a step-wise
process. In either case, the barrier for phenyl acrylate formation is very high, computed
to be about 80 kcal/mol from the zwitterion, and 90 - 100 kcal/mol from the canonical
structure.
Although loss of CO2 from the amino acid is not observed in this work, it has
been reported previously, and therefore we have carried out calculations to determine
93
why it does not occur for PheSO3. Decarboxylation of the zwitterionic structure of Phe
is calculated to occur without a barrier in excess of the dissociation energy, such that the
net barrier for the reaction is determined by the energies of the dissociation products.
Therefore, loss of CO2 has a barrier of more than 60 kcal/mol. As shown in Figure 5.4,
loss of CO2 would necessarily lead to formation of the zwitterionic product (the
ammonium-ylide) and not the phenethyl amine. Although formation of the amine is
exothermic, Alexandrova and Jorgensen63 have noted that the barrier for proton shift in
the ylide is formally symmetry forbidden and is therefore expected to have a very high
barrier. Consequently, the formation of the amine from glycine is essentially a
sequential, two-step process consisting of decarboxylation followed by proton transfer,
with a proton transfer barrier of approximately 45 kcal/mol for the ylide in aqueous
solution.63 The barrier for proton shift in the ammonium-ylide formed upon
decarboxylation of Phe in the gas phase is calculated to be 25.6 kcal/mol, such that the
overall barrier for formation of the amine upon decarboxylation of Phe is calculated to be
86.4 kcal/mol. The higher barrier for proton shift computed for glycine is likely due to
solvent stabilization of the ammonium-ylide. Interestingly, Alexandrova and Jorgensen
also calculated a slight (~8 kcal/mol) barrier in excess of the dissociation energy for the
decarboxylation step. However, the presence of the excess barrier is also likely a
consequence of explicitly including solvation in the QM/MM approach, and would not be
expected in a gas-phase calculation.64 The barriers for decarboxylation obtained for
glycine by Alexandrova and Jorgensen63 and for Phe in this work are significantly higher
than what has been reported previously for the decarboxylation of canonical N,N-
dimethyl- or N-phenylglycine.20,21,65 However, the ~42 kcal/mol barriers suggested in the
94
previously studies do not seem reasonable because they are lower than the energies
required for decarboxylation, which we calculate to be 52.3 kcal/mol and 59.6 kcal/mol
for dimethylglycine and N-phenylglycine, respectively. Considering that decarboxylation
leads initially to the ammonium-ylide, and that rearrangement of the ylide to the amine
occurs in a second step and is formally symmetry forbidden, decarboxylation should not
occur with an activation energy less than that required for formation of the ammonium-
ylide.
In summary, although loss of CO2 from the amino acid can occur to form the
ammonium-ylide, formation of the α-lactone is predicted to be energetically preferred by
about 20 kcal/mol. Consequently, that is the only process that is observed for PheSO3.
5.6.2 Dissociation of the α-Lactone
In this study, we have also been able to investigate the dissociation of the α-
lactone, αLSO3. α-Lactones are highly reactive molecules that have been proposed as
short lived intermediates in a variety of chemical reactions, often involving α-substituted
carboxylates50,54-60 or α,β-unsaturated acids,66,67 in the photolysis of cyclic peroxides47,68-
71 and α-halocarboxylic acids,72 in the oxidation of ketenes,73 or in the gas-phase
dissociation of α-substituted carboxylic24-29 or in the decomposition of amino acids.23,74
They have also been proposed as intermediates in atmospheric oxidation reactions75,76 in
mass spectrometry,77-80 and as products of the reaction of carbenes with CO2.81-85 Despite
being highly reactive, α-lactones have been investigated spectroscopically by using
matrix isolation,82-84 gas-phase86 and solution-phase time-resolved IR.85 There has long
been discussion regarding the electronic structure of α-lactones, and whether they are best
95
considered to be cyclic, canonical structures56,58-60 or zwitterionic,55 although those
discussions generally relate to the structure in solution. The calculated structures of gas-
phase α-lactones have exceptionally long (~1.55 Å) and weak87 Cα-O bonds, and even in
the gas phase there is considerable ionic character.87
α-Lactones are highly reactive, and react rapidly by polymerization, by
nucleophilic addition, or by decomposition.88 Although an early photolysis study
reported dissociation by loss of CO and CO2,47 more recent studies of α-substituted
carboxylic acid pyrolyses that proceed through α-lactone intermediates24,25,30-36 have
reported dissociation only by loss of CO. In this work, we observe loss of both CO and
CO2 from the α-lactone, in similar amounts (if anything, CO2 loss is slightly favored). As
described above, loss of CO occurs to form the aldehyde, as expected, whereas loss of
CO2 results in the formation of the styrene (eq 5.10).
Computed potential energy surfaces for the decomposition channels are shown in
Figure 5.5. The barriers for CO loss from the α-lactone derived from PheSO3 and Phe
are predicted to be about 30 kcal/mol, similar to that predicted for alkyl-substituted α-
lactones.52 Determining the barrier for styrene formation is more difficult. The
experimental observation that CO and CO2 are formed in similar indicates that the
barriers for the two processes are similar. Therefore, a stepwise mechanism consisting of
decarboxylation to form the carbene followed by hydrogen shift likely has a barrier that is
too high to compete with CO loss (see Figure 5.5). Considering that decarboxylation of
β-lactones results directly in the formation of the olefin and occurs with moderate energy
barriers89,90 we explored the possibility of a mechanism involving the dyotropic shift91-94
to the β-lactone95 followed by decarboxylation (eq 5. 13a). However, the search for the
96
transition state for α- to β-lactone rearrangement did not give a structure for a dyotropic
process, as shown in 10a, but instead gave a structure that consists of 1,2-hydrogen shift
without assistance of the carboxylate anion, as shown in eq 5.13b. The structure shown
in eq 5.13b was confirmed to be a saddle point by vibrational analysis, and an intrinsic
reaction coordinate calculation (IRC)96,97 finds that it is a transition state that connects the
α-lactone with the styrene and CO2.
Figure 5.5. Calculated energetics for the dissociation of αLSO3 (X = SO3
) and the non-sulfonated derivative (X = H). Values correspond to electronic energies and geometries calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of theory, unless indicated a)
not a stable geometry; corresponds to the geometry of the lactone with the CO2 removed.
97
The 1,2-hydrogen shift mechanism shown in eq 5.13b is facilitated by the high
degree of polarization expected for the α-lactone (eq 5.14),87 and by the fact that the β-
cationic carboxylate (β(+)-CO2) that would result from hydride transfer is unstable with
respect to decarboxylation in the gas phase. The coupling of hydrogen migration and
leaving-group loss is analogous to what has been proposed for the Schmidt reaction,94
and has been observed previously in reactions such as the elimination of protonated
ethers98,99 and acetates.100
The computed barriers for decarboxylation of the α-lactone are 32.1 kcal/mol for
αLSO3, and 42.1 kcal/mol for the non-ionic system, indicating that the sulfonate is
98
having a large effect on the stability of the transition state. Because the charge of the
sulfonate group does not delocalize into the π-system of the ring via conjugation, the
stabilization of the transition state is likely an inductive effect that stabilizes the
formation of the benzylic cation. The effect would be expected to be stronger for an
ionic group that is conjugated with the aromatic ring, such as an O group of a phenoxide
(eq 5.15), which accounts for why the phenoxide ion obtained from deprotonating
tyrosine loses NH3 and CO2 as the main dissociation channel, and an M-NH3-CO ion is
not observed at all.53
OOH
H H
OOH
H H+
_
(5.15)
HH
CO2
_
H
O O_ _
O
The calculated barriers for decarboxylation and decarbonylation of the ionic and
non-ionic substrates are consistent with the experimentally observed results. The large
difference in barrier heights for the non-ionic lactone accounts for why loss of CO is
generally the only observed channel in pyrolysis experiments. However, for αLSO3, the
computed barriers for loss of CO and CO2 are very similar, which accounts for why the
products are observed in nearly equal amounts. As shown in eq 5.15, the resonant
interaction between the charge site and the benzylic site in deprotonated tyrosine is
expected to strongly favor the CO2 loss channel. Finally, preliminary results with para-
99
trimethylammonium-substituted Phe find that the corresponding α-lactone dissociates
only by loss of CO, consistent with electrostatic destabilization of the benzylic cation.
5.7 Conclusions
By using an ion with the charged-moiety isolated from an amino acid, we have
been able to utilize mass spectrometry to investigate the chemical properties of a gas-
phase amino acid. In this work, we have characterized the decomposition pathways for a
phenylalanine derivative. The only observed pathway is loss of ammonia, to form an α-
lactone. This reaction is similar to what has been observed previously for pyrolysis of N-
substituted alanines23 and other α-substituted carboxylic acids,24-36 but is inconsistent
with what has been reported for pyrolysis of glycine derivatives.20-22 The main difference
between the studies that find deamination23 (including this work) and those that find
decarboxylation to occur20-22 is our work and that of Al-Awadi et al.23 involves a direct
investigation of the gaseous amino acid, whereas Chuchani and co-workers have tried to
generate the amino acid in situ by pyrolysis of the corresponding ethyl esters.
Considering that the computational results in this work and by others63 have shown that
decarboxylation of gaseous amino acids to form the amine has a prohibitively high
barrier, the products that correspond to decarboxylation of the amine are likely formed
via a pathway not involving the amino acid.
The α-lactone that is formed from the amino acid in this work undergoes
dissociation by loss of CO and by loss of CO2. Although loss of CO is expected, the loss
of CO2 has generally not been reported in reactions involving α-lactones. However,
given the calculated relative barriers for CO and CO2 loss, decarboxylation should occur,
100
to some extent, in α-lactones containing a β-hydrogen that can shift during lactone ring
opening, similar to the reaction shown in eq 5.13b. It may be that the formation of the
benzylic cation may facilitate the hydride shift reaction, although decarboxylation has
been observed for a completely aliphatic derivative (eq 5.9).47 The results of this work
and that reported previously for deprotonated tyrosine show that stabilization of the β-
cation can affect the branching ratio for CO vs CO2 loss. Therefore, just as anionic
stabilization of the transition state favors CO2, the presence of a cationic group would be
expected to disfavor decarboxylation, and favor loss of CO, which is what is observed in
preliminary studies.
Finally, the results of this study show that mass spectrometry can be used to
investigate the properties of canonical amino acids. Whereas this is a new approach for
the investigation of gas-phase amino acids, the use of an inert, remote charge to
investigate neutral chemistry is not new and is similar, in principle, to the distonic ion
approach used by Kenttämaa and co-workers101 to examine the reactivity of aromatic
radicals. In the same way, it should be possible to use mass spectrometry to investigate
further the reactivity of gas-phase amino acids, and investigate the effects of structure and
solvation.
101
5.8 References
(1) Kuan, Y.-J.; Charnley, S. B.; Huang, H.-C.; Tseng, W.-L.; Kisiel, Z. Astrophys. J. 2003, 593, 848.
(2) Snyder, L. E.; Lovas, F. J.; Hollis, J. M.; Friedel, D. N.; Jewell, P. R.; Remijan, A.; Ilyushin, V. V.; Alekseev, E. A.; Dyubko, S. F. Astrophys. J. 2005, 619, 914.
(3) Johansson, A. C. V.; Lindahl, E. J. Chem. Phys. 2009, 130, 185101.
(4) Chapo, C. J.; Paul, J. B.; Provencal, R. A.; Roth, K.; Saykally, R. J. J. Am. Chem. Soc 1998, 120, 12956.
(5) Julian, R. R.; Jarrold, M. F. J. Phys. Chem. A 2004, 108, 10861.
(6) Jensen, J. H.; Gordon, M. S. J. Am. Chem. Soc. 1995, 117, 8159.
(7) Kassab, E.; Langlet, J.; Evleth, E.; Akacem, Y. J. Mol. Struct. 2000, 531, 267.
(8) Jarrold, M. F. Annu. Rev. Phys. Chem. 2000, 51, 179.
(9) Zwier, T. S. J. Phys. Chem. A 2006, 110, 4133.
(10) Bakker, J. M.; Aleese, L. M.; Meijer, G.; Helden, G. v. Phys. Rev. Lett. 2003, 91, 203003.
(11) Schermann, J.-P. Spectroscopy and Modelling of the Biomolecular Building Blocks; Elsevier: Netherland, 2008.
(12) Hu, Y.; Bernstein, E. R. J. Chem. Phys. 2008, 128, 164311/1.
(13) Hu, Y.; Bernstein, E. R. J. Phys. Chem. A 2009, 113, 8454.
(14) Antoine, R.; Dugourd, P. Phys. Chem. Chem. Phys. 2011, 13, 16494.
(15) Gao, Y. K.; Traeger, F.; Kotsis, K.; Staemmler, V. Phys. Chem. Chem. Phys. 2011, 13, 10709.
(16) Jochims, H.-W.; Schwell, M.; Chotin, J.-L.; Clemino, M.; Dulieu, F.; Baumgartel, H.; Leach, S. Chem. Phys. 2004, 298, 279.
(17) Adams, J. Mass Spectrom. Rev. 1990, 9, 141.
(18) Cheng, C.; Gross, M. L. Mass Spectrom. Rev. 2000, 19, 398.
(19) Adams, J.; Gross, M. L. J. Am. Chem. Soc. 1989, 111, 435.
102
(20) Ensuncho, A.; Lafont, M. J.; Rotinov, A.; Dominguez, R. M.; Herize, A.; Quijano, J.; Chuchani, G. Int. J. Chem. Kinet. 2001, 33, 465.
(21) Dominguez, R. M.; Tosta, M.; Chuchani, G. J. Phys. Org. Chem. 2003, 16, 869.
(22) Tosta, M.; Oliveros, J. C.; Mora, J. R.; Cordova, T.; Chuchani, G. J. Phys. Chem. A 2010, 114, 2483.
(23) Al-Awadi, S. A.; Abdallah, M. R.; Hasan, M. A.; Al-Awadi, N. A. Tetrahedron 2004, 60, 3045.
(24) Al-Awadi, N. A.; Kumar, A.; Chuchani, G.; Herize, A. Int. J. Chem. Kinet. 2001, 33, 612.
(25) Chuchani, G.; Dominguez, R. M.; Rotinov, A.; Martin, I. J. Phys. Org. Chem. 1999, 12, 612.
(26) Domingo, L. R.; Picher, M. T.; Andres, J.; Safont, V. S.; Chuchani, G. Chem. Phys. Lett. 1997, 274, 422.
(27) Domingo, L. R.; Picher, M. T.; Safont, V. S.; Andres, J.; Chuchani, G. J. Phys. Chem. A 1999, 103, 3935.
(28) Rotinov, A.; Chuchani, G.; Andre, J.; Domingo, L. R.; Safont, V. S. Chem. Phys. 1999, 246, 1.
(29) Safont, V. S.; Moliner, V.; Andres, J.; Domingo, L. R. J. Phys. Chem. A 1997, 101, 1859.
(30) Chuchani, G.; Dominguez, R. M. Int. J. Chem. Kinet. 1995, 27, 85.
(31) Chuchani, G.; Dominguez, R. M. Int. J. Chem. Kinet. 1999, 31, 725.
(32) Chuchani, G.; Dominguez, R. M.; Rotinov, A. Int. J. Chem. Kinet. 1991, 23, 779.
(33) Chuchani, G.; Martin, I.; Rotinov, A. Int. J. Chem. Kinet. 1995, 27, 849.
(34) Chuchani, G.; Martin, I.; Rotinov, A.; Dominguez, R. M. J. Phys. Org. Chem. 1993, 6, 54.
(35) Chuchani, G.; Rotinov, A. Int. J. Chem. Kinet. 1989, 21, 367.
(36) Chuchani, G.; Rotinov, A.; Dominguez, R. M. J. Phys. Org. Chem. 1996, 9, 787.
103
(37) Version 9.6 ed.; Schrödinger, LLC: New York, NY, 2009.
(38) Kong, J.; White, C. A.; Krylov, A. I.; Sherrill, D.; Adamson, R. D.; Furlani, T. R.; Lee, M. S.; Lee, A. M.; Gwaltney, S. R.; Adams, T. R.; Ochsenfeld, C.; Gilbert, A. T. B.; Kedziora, G. S.; Rassolov, V. A.; Maurice, D. R.; Nair, N.; Shao, Y.; Besley, N. A.; Maslen, P. E.; Dombroski, J. P.; Daschel, H.; Zhang, W.; Korambath, P. P.; Baker, J.; Byrd, E. F. C.; Voorhis, T. V.; Oumi, M.; Hirata, S.; Hsu, C.-P.; N.Ishikawa; Florian, J.; A.Warshel; Johnson, B. G.; Gill, P. M. W.; M.Head-Gordon; Pople, J. A. J. Comput. Chem. 2000, 21, 1532.
(39) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; G. E. Scuseria; Robb, M. A.; Cheeseman, J. R.; J. A. Montgomery, J.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.; Gaussian, Inc.: Pittsburgh, PA, 2003.
(40) Bartmess, J. E. 2012.
(41) Nha Tran, T. T.; Wang, T.; Hack, S.; Bowie, J. H. Rapid Commun. Mass Spectrom. 2013, 27, 1135.
(42) Tian, Z.; Pawlow, A.; Poutsma, J. C.; Kass, S. R. J. Am. Chem. Soc 2007, 129, 5403.
(43) Tian, Z.; Wang, X.-B.; Wang, L.-S.; Kass, S. R. J. Am. Chem. Soc. 2009, 131, 1174.
(44) Eckersley, M.; Bowie, J. H.; Hayes, R. N. Int. J. Mass Spectrom. Ion Processes 1989, 93, 199.
(45) Choi, S.-S.; Kim, O.-B. Int. J. Mass Spectrom. 2013, 338, 17.
(46) Couldwell, A. M.; Thomas, M. C.; Mitchell, T. W.; Hulbert, A. J.; Blanksby, S. J. Rapid Commun. Mass Spectrom. 2005, 19, 2295.
(47) Adam, W.; Rucktaeschel, R. J. Org. Chem. 1978, 43, 3886.
(48) Baker, M.; Gabryelski, W. Int. J. Mass Spectrom. 2007, 262, 128.
104
(49) Choi, T. S.; Ko, J. Y.; Heo, S. W.; Ko, Y. H.; Kim, K.; Kim, H. I. J. Am. Soc. Mass Spectrom. 2012, 23, 1786.
(50) Graul, S. T.; Squires, R. R. Int. J. Mass Spectrom. Ion Processes 1990, 100, 785.
(51) Rijs, N. J.; O'Hair, R. A. J. Dalton Trans. 2012, 41, 3395.
(52) Domingo, L. R.; Andres, J.; Moliner, V.; Safont, V. S. J. Am. Chem. Soc. 1997, 119, 6415.
(53) Tian, Z.; Kass, S. R. J. Am. Chem. Soc. 2008, 130, 10842.
(54) Bean, C. M.; Kenyon, J.; Phillips, H. J. Chem. Soc. 1936, 303.
(55) Cowdrey, W. A.; Hughes, E. D.; Ingold, C. K.; Masterman, S.; Scott, A. D. J. Chem. Soc. 1937, 1252.
(56) Grunwald, E.; Winstein, S. J. Am. Chem. Soc. 1948, 70, 841.
(57) Strijtveen, B.; Kellogg, R. M. Recl. Trav. Chim. Pays-Bas 1987, 106, 539.
(58) Winstein, S. J. Am. Chem. Soc. 1939, 61, 1635.
(59) Winstein, S.; Henderson, R. B. J. Am. Chem. Soc. 1943, 65, 2196.
(60) Winstein, S.; Lucas, H. J. J. Am. Chem. Soc. 1939, 61, 1576.
(61) Antolovic, D.; Shiner, V. J.; Davidson, E. R. J. Am. Chem. Soc. 1988, 110, 1375.
(62) Hamada, Y.; Takeo, H. Appl. Spectrosc. Rev. 1992, 27, 289.
(63) Alexandrova, A. N.; Jorgensen, W. L. J. Phys. Chem. B 2011, 115, 13624.
(64) Alexandrova, A. N., personal communication.
(65) Perez, J. R.; Lorono, M.; Dominguez, R. M.; Cordova, T.; Chuchani, G. J. Phys. Org. Chem. 2008, 21, 402.
(66) Pirinccioglu, N.; Robinson, J. J.; Mahon, M. F.; Buchanan, J. G.; Williams, I. H. Org. Biomol. Chem. 2007, 5, 4001.
(67) Niwayama, S.; Noguchi, H.; Ohno, M.; Kobayashi, S. Tetrahedron Lett. 1993, 34, 665.
(68) Adam, W.; Liu, J.-C.; Rodriguez, O. J. Org. Chem. 1973, 38, 2269.
105
(69) Adam, W.; Rucktaeschel, R. J. Amer. Chem. Soc. 1971, 93, 557.
(70) Chapman, O. L.; Wojtkowski, P. W.; Adam, W.; Rodriguez, O.; Rucktaeschel, R. J. Amer. Chem. Soc. 1972, 94, 1365.
(71) Adam, W.; Blancafort, L. J. Org. Chem. 1996, 61, 8432.
(72) Nishino, S.; Nakata, M. J. Mol. Struct. 2008, 875, 520.
(73) Schmittel, M.; von, S. H. Liebigs Ann. 1995, 1815.
(74) Kenyon, J.; Phillips, H. Trans. Faraday Soc. 1930, 26, 451.
(75) Carr, S. A.; Glowacki, D. R.; Liang, C.-H.; Baeza-Romero, M. T.; Blitz, M. A.; Pilling, M. J.; Seakins, P. W. J. Phys. Chem. A 2011, 115, 1069.
(76) Kjaergaard, H. G.; Knap, H. C.; Oernsoe, K. B.; Joergensen, S.; Crounse, J. D.; Paulot, F.; Wennberg, P. O. J. Phys. Chem. A 2012, 116, 5763.
(77) Schroder, D.; Goldberg, N.; Zummack, W.; Schwarz, H.; Poutsma, J. C.; Squires, r. R. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 71.
(78) Lam, A. K. Y.; O'Hair, R. A. J. Rapid Commun. Mass Spectrom. 2010, 24, 1779.
(79) Wyer, J. A.; Feketeova, L.; Brondsted, N. S.; O'Hair, R. A. J. Phys. Chem. Chem. Phys. 2009, 11, 8752.
(80) Armentrout, P. B.; Ye, S. J.; Gabriel, A.; Moision, R. M. J. Phys. Chem. B 2010, 114, 3938.
(81) Kistiakowsky, G. B.; Sauer, K. J. Am. Chem. Soc. 1958, 80, 1066.
(82) Milligan, D. E.; Jacox, M. E. J. Chem. Phys. 1962, 36, 2911.
(83) Wierlacher, S.; Sander, W.; Liu, M. T. H. J. Org. Chem. 1992, 57, 1051.
(84) Sander, W. W. J. Org. Chem. 1989, 54, 4265.
(85) Showalter, B. M.; Toscano, J. P. J. Phys. Org. Chem. 2004, 17, 743.
(86) Chen, S.-Y.; Lee, Y.-P. J. Chem. Phys. 2010, 132, 114303/1.
(87) Ruggiero, G. D.; Williams, I. H. J. Chem. Soc., Perkin Trans. 2 2001, 733.
(88) L'Abbé, G. Angew. Chem., Int. Ed. Eng. 1980, 19, 276.
106
(89) Morao, I.; Lecea, B.; Arrieta, A.; Cossio, F. P. J. Am. Chem. Soc. 1997, 119, 816.
(90) Ocampo, R.; Dolbier, W. R., Jr.; Bartberger, M. D.; Paredes, R. J. Org. Chem. 1997, 62, 109.
(91) Reetz, M. T. Angew. Chem., Int. Ed. Engl. 1972, 11, 130.
(92) Reetz, M. T. Angew. Chem., Int. Ed. Engl. 1972, 11, 129.
(93) Fernandez, I.; Cossio, F. P.; Sierra, M. A. Chem. Rev. (Washington, DC, U. S.) 2009, 109, 6687.
(94) Gutierrez, O.; Tantillo, D. J. J. Org. Chem. 2012, 77, 8845.
(95) Buchanan, J. G.; Ruggiero, G. D.; Williams, I. H. Org. Biomol. Chem. 2008, 6, 66.
(96) Gonzalez, C.; Schlegel, H. B. J. Chem. Phys. 1989, 90, 2154.
(97) Gonzalez, C.; Schlegel, H. B. J. Phys. Chem. 1990, 94, 5523.
(98) Ben, A. J.; Karni, M.; Apeloig, Y.; Mandelbaum, A. Int. J. Mass Spectrom. 2003, 228, 297.
(99) Morlender-Vais, N.; Mandelbaum, A. J. Mass Spectrom. 1997, 32, 1124.
(100) Kuzmenkov, I.; Etinger, A.; Mandelbaum, A. J. Mass Spectrom. 1999, 34, 797.
(101) Smith, R. L.; Thoen, K. K.; Nousiainen, J. J.; Nelson, E. D.; Kenttamaa, H. I. J. Am. Chem. Soc. 1996, 118, 8669.
107
CHAPTER SIX: EFFECT OF CHARGE POLARITY ON AMINO ACID DISSOCIATION
6.1 Introduction
The structures, properties, and reactivity of amino acids are well-characterized in
condense phase 1-4 and in solution. 5-7 However, owing to their lack of volatility, much
less is known about the structure and reactivity of amino acids in the gaseous state. 8,9
Mass spectrometry has been used to study the gas phase properties of amino acids.10-13
However, in these studies, the amino acids are typically ionized in some way, such as via
protonation 11,12 or metallation to create positively-charged ions, or by deprotonation 11 to
form negatively-charged ions. Consequently, the properties of ionized amino acids
investigated by using mass spectrometry are generally not comparable to those of the
“neutral” (zwitterionic or canonical) amino acids.13
Recently, we reported the charge-remote fragmentation14,15 of para-sulfonated
phenylalanine anion, PheSO3, using mass spectrometry.13 We discovered PheSO3
dissociates only by loss of NH3 to make a product assigned to be an α-lactone. Here, we
report the findings on charge-remote fragmentation of N,N,N-trimethyl ammonium
phenylalanine, PheNMe3+, and compared with PheSO3 . Same experimental and
computational methods as explained in Chapter 5 for PheSO3 were used in this study.
108
6.2 Synthesis
All N,N,N-trimethyl ammonium derivatives were synthesized by methylation of
aniline. Boc protected samples were used as the starting materials. For example,
PhNMe3+Cl was synthesized using Boc-4-aminophenylalanine as a starting material.
0.06 g of Boc-4-aminophenylalanine and 3 equivalent of 2, 6-lutidine were dissolved in 2
ml of DMF, and 2.5 ml of CH3I was added. The solution was stirred overnight in room
temperature. Excess solvent and CH3I were then evaporated under vacuum. The
precipitated salt was filtered and the Boc group was removed by treating with an excess,
~2.5 ml, of trifluoroacetic acid (TFA). Excess TFA was removed by evaporation under
vacuum and the product was studied in mass spectrometry.
6.3 Results and Discussion
Calculations have been carried out on the structure of PheNMe3+ in the gas phase.
Figure 6.1 shows the 7 lowest energy canonical conformation of PheNMe3+ and the
relative energies of each stationary point optimized at the B3LYP/6-31+G* level of
theory. The conformation preferences in PheNMe3+ are based on the orientation of
carboxylic acid, where cis orientation of COOH is more favorable than trans orientation.
109
However, the conformation preferences in PheSO3 are based on the relative position of
carboxylic acid with aromatic ring, where anti is more favorable than gauche (Chapter 5).
The conformational preference is not expected to affect the gas-phase properties of these
charge-remote amino acids since all structures are within 5 kcal/mol.
Figure 6.1: The 7 low energy conformations of PheNMe3+. Relative energies are shown in kcal/mol
6.3.1 Dissociation of PheNMe3+
Upon collision-induced dissociation, the PheNMe3+ with m/z 206 losses the
neutrals of masses 17, 44, 45, 59, 61, 74, 76, 87, 88 and 89 Dalton (Da), as shown in
Figure 6.2a. Other than the 17 and 45 Da losses, the other fragmentation pathways were
not observed on fragmentation of PheSO3 (Chapter 5).13 The techniques like H/D
0 0.2 0.9 1 A B C D
1 1 2 E F G
110
exchange of labile protons (d-labeled compounds), authentic sample comparison and
tandem MS are implemented to help determine the structures of the neutrals lost and the
product ions formed.
Figure 6.2. CID spectra of a) PheNMe3+ with m/z 223, b) d3- PheNMe3+ with m/z 226
m/z
130 140 150 160 170 180 190 200 210 220 2300.0
0.2
0.4
0.6
0.8
1.0
1.2
130 140 150 160 170 180 190 200 210 220 230
Rel
Int
0.0
0.2
0.4
0.6
0.8
1.0
1.2
X 10
X 10
206
223
179
178
164
162
149
147
136
134 135
206
226
182
178
167
162
149
147
137
134 138
207
a
b
111
6.3.1.1 Neutral Loss of Mass 17 Da and Formation of m/z 206 ion
Two neutrals, NH3 and OH, are possible fragments of mass 17 Da that can be lost
from PheNMe3+. The CID spectrum of the d3-PheNMe3+ with m/z 226 shown in Figure
6.2b does not have a peak at m/z 208 accounting for the loss of the OD radical. Therefore
the loss of hydroxy radical can be ruled out confirming the loss of ammonia. The
ammonia loss is a common fragment present in the CID of protonated,11,12 deprotonated11
and charge-remote13 phenylalanine.
Although the structure of the deamination product is not known, the most likely
products to be formed are either the cinnamic acid or an α-lactone derivatives (Chapter
5.4.1). The deamination product observed on dissociation of PheSO3 was deduced to be
the α-lactone based on its dissociation behavior and by ruling out possible alternatives.13
However, the recent ion spectroscopy shows the product obtained upon dissociation of
the sulfonated phenylalanine is an α-lactone confirming the assignment made previously
based on indirect evidence.
Upon CID, the [PheNMe3 –NH3]+ ion with m/z 206 losses neutrals of masses 15,
42, 44 and 46 Da. The molecular formulas of CH3, CO2 and CO2H2 can be attributed to
the masses 15, 44 and 46 Da respectively but that for neutral of 42 Da could not be
assigned. However, the neutral CO loss, a well-known decomposition pathway for α-
lactone, is not observed in the CID of the deamination product 13,16-20 suggesting the
deamination product is most likely the cinnamic acid derivative (1), Figure 6.7 (a).
A comparison of the CID spectra of [PheNMe3 –NH3]+ and the authentic 1 with
m/z 206, measured with a normalized collision energy of 35%, are shown in Figure 6.3.
The CID of the authentic 1 forms a single product ion at m/z 191, which probably is due
112
to loss of CH3. Even though the other peaks present in CID spectrum of [PheNMe3 –
NH3]+ are not observed on the CID spectrum of 1, the fact both spectra in Figure 6.3 have
m/z 191 as the base peak suggest the similarity on the structures of the [PheNMe3 –
NH3]+ and 1.
Figure 6.3. CID spectra of m/z 206 obtained from a) CID of PheNMe3+, b) authentic cinnamic acid (1)
206
191
191
206
164
162160
a
b
113
This result is also consistent with the deuterated experiment. Upon CID, the d3-
PheNMe3+ with m/z 226 losses neutrals of 19 and 20 Da which are possibly ND2H and
ND3 12 loss with a proton coming from the ethylene backbone or the carboxylic acid
respectively13 to form 1. Formation of the cinnamic acid has been reported as a minor
product for pyrolysis21,22 and thermolysis23 of phenylalanine in the condensed phase.
6.3.1.2 Neutral Loss of Mass 44 Da and Formation of m/z 179 ion
Two neutral molecules, CO2 and NMe2, are possible fragments with mass 44 Da
that can be lost from PheNMe3+. The CID spectrum of the [PheNMe3 – 44]+ matches
with the authentic phenylethanamine, 2, (Figure 6.4 a and b). This result confirms CO2 is
lost upon CID of PheNMe3+ and [PheNMe3 –CO2]+ ion with m/z 179 is 2, Figure 6.7 (b).
This result is also consistent with d-labeled experiment since CID of d3-PheNMe3+ loses
neutral of mass 44 Da as well, Figure 6.2 b).
The product ions formed upon CID of m/z 2 are also present in CID spectrum of
PheNMe3+. Thus, the CID spectrum of 2 and d2-2 shown in Figure 6.4 b and c are
compared to get further insight on fragmentation pathways. The product ions with m/z
164(166), 162(162), 135(136), and 134(134) formed upon CID of 2 (d2-2) with m/z
179(181) are due to the neutral loss of mass 15(15), 17(19), 44(45), and 47(45) Da
(number in bracket for deuterated sample).
114
Figure 6.4. CID spectra of a) m/z 179 obtained from CID of PheNMe3+, b) 2 with m/z 179, c) d2-2 with m/z 181
179
179
181
166
136
134
164
164
135
134
135
134
a
b
c
115
The neutral loss of 15(15) Da is likely CH3 radical from the ammonium group to
form [2 – CH3]+ with m/z 164(166). The CID of m/z 164(166) losses neutrals with masses
29(30) and 30(32) Da, probably to form [2 – CH3 - NHCH2]+ with m/z 135(136) and [2 –
CH3 - NH2CH2]+ with m/z 134(134), respectively. However, the CID spectrum of
[PheNMe3 – 59]+ with m/z 164 has product ions at m/z 149, 147, 135, 134, 136, 121 and
120, which are more than CID spectrum of [2-CH3] with m/z 164, Figure 6.5. The
common base peaks in both spectra suggest that the m/z 164 is mostly [PheNMe3 –CO2 -
CH3]+ with some impurities of other isobaric ion.
Figure 6.5. CID spectra of m/z 164 obtained from a) 2, b) PheNMe3+
m/z
120 130 140 150 160 170
Rel
Int
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0 a
b
164
164
149147
136134
135
135
120
121
a
b
116
Therefore, the m/z 135 and 134 seen in CID spectrum of PheNMe3+ are probably
[PheNMe3 –CO2 – CH3 - NHCH2]+ and [PheNMe3 –CO2 – CH3 - NH2CH2]+, Figure 6.7
(b.2.1) and (b.2.2), respectively.
The neutral of 17(19) Da with mass difference of 2 indicates both labile protons
are lost in neutral, probably as NH3 to produce [2 – NH3]+ with m/z 162(162). The CID of
[2 –NH3]+ ion loses neutral of mass 15 Da, probably CH3 neutral from ammonium site, to
produce [2 –NH3 – CH3]+ with m/z 149. Therefore, the m/z 164 and 149 seen in CID
spectrum of PheNMe3+ are probably [PheNMe3 –CO2 – NH3]+ and [PheNMe3 – CO2 –
NH3 – CH3]+, Figure 6.7 (b.1) and (b.1.1), respectively.
The decarboxylation is the main fragmentation pathway of PheNMe3+ since the
base peak at m/z 179 in CID spectrum of PheNMe3+ is due to decarboxylation. In
addition, decarboxylation leads to several other product ions like m/z 164, 162, 147, 135,
and 134 present in CID spectrum of PheNMe3+. Decarboxylation has been reported as a
major product for pyrolysis21,22 and thermolysis23 of phenylalanine in the condensed
phase as well. However, CO2 fragmentation pathway is not observed in collision-induced
fragmentation of protonated, deprotonated or charge-remote phenylalanine.
Since CID is a kinetic process, comparing the barrier a fragmentation pathways
might provide further insight on why this fragmentation is observed in one case and not
in other. Figure 6.6 shows the potential energy surface for the decarboxylation of
PheSO3, PheNMe3+ and Phe. The decarboxylation to form zwitterionic products (the
ammonium-ylide) is slightly favorable for PheNMe3+ but the experimental evidence
suggests decarboxylation leads to phenylethyl amine. Formation of the amine is
exothermic, but the barrier for proton shift in the ylide is symmetry forbidden and is
117
therefore expected to have a very high barrier. The barrier for proton shift in the
ammonium-ylide formed upon decarboxylation of Phe in the gas phase is calculated to be
25.6 kcal/mol, allowing the overall barrier for formation of the amine upon
decarboxylation of Phe to be calculated at 86.4 kcal/mol. The potential energy diagram
does not provide any testiment on why decarboxylation is preferred only in PheNMe3+.
X
CO2H
NH2
X
NH3+
_
X
NH2
X
NH3+
_
(86.4)[75.8]
X
NH2
H
+ CO2
+ CO2
+ CO2
0.02.2
(-3.6)
8.9
(13.5a)
[10.4]
63.7(60.8)[49.8]
Values in kcal/molX = SO3(X = H)[X=NMe3
+]
_
O
O
Figure 6.6. PES for decarboxylation of PheNMe3+, PheSO3 and Phe. The barriers and the molecular energy were calculated at the MP2/6-311+G**//B3LYP/6-31+G* level of
theory.
118
6.3.1.3 Neutral Loss of Mass 45 Da and Formation of m/z 178 ion
Two neutral molecules, CO2H and CONH3, are the possible fragments with mass
45 Da that can be lost from PheNMe3+. The CID spectrum of d3-PheNMe3+ shown in
Figure 6.2 b) depicts all three labile protons are lost to yield m/z 178 suggesting CONH3
is the neutral lost. The neutral loss of 45 Da was observed for the CID of PheSO3 as
well, which was loss of CO from deamination product. Since the CID [PheNMe3 - NH3] +
with did not lose CO, Figure 6.3 a), CONH3 is probably lost as a single fragment to
produce [PheNMe3 – CONH3]+ with m/z 178, Figure 6.7 (c).
6.3.1.4 Neutral Loss of Mass 74 Da and Formation of m/z 149 ion
C2O2NH4 is the only possible fragment with mass 74 Da that can be lost form
PheNMe3+. This neutral fragment is likely CH(NH2)COOH that result in formation of
the benzyl radical ion, [PheNMe3 – CH(NH2)COOH]+, with m/z 149, Figure 6.7 (d).
This result is consistent with CID of d3-PheNMe3+, where all labile protons are lost to
form m/z 149 product, Figure 6.2 b). Upon CID, [PheNMe3 – CH(NH2)COOH]+ with m/z
149 losses neutral of mass 15 Da, which probably is loss of CH3. Therefore, the m/z 134
seen in CID spectrum of PheNMe3+ is assigned as [PheNMe3 – CH(NH2)COOH – CH3]+,
Figure 6.7 (d.1).
6.3.1.5 Neutral Loss of Mass 87 Da and Formation of m/z 136 ion
The neutral with mass 87 Da lost from PheNMe3+ is likely C3O2NH5. The ionic
product has a formula that matches trimethylanilinum, [PheNMe3 – Ala + H]+ with m/z
136, Figure 6.7 (e). The CID spectrum of d3-PheNMe3+ with m/z 226 contains peaks at
119
m/z 136 and 137, Figure 6.2 b), suggesting the proton donated by alanine could be the
labile or not labile hydrogen.
6.4 Conclusion
Upon collison induded dissociation, PheNMe3+ m/z 223 produces the fragment
ions with m/z 206, 179, 178, 164, 162, 149, 147, 136, 135 and 134. The proposed
fragmentation pathways of PheNMe3+ are summarized in Figure 6.7.
The product ions with m/z 206, 179, 178, 149 and 136 are identified as primary
fragmentation pathways. The product ions with m/z 164 and 162 are secondary
fragmentation pathways and those with m/z 147, 135 and 134 are tertiary fragmentation
pathways. Far more fragmentation pathways are found for PheNMe3+ than for PheSO3-.
In PheSO3, deammination was the only primary fragmentation pathway, whereas
PheNMe3+ has 4 additional pathways. The neutrals loss of mass 45 and 74 Da proposed
as the primary fragmentation pathway on this study were observed as the secondary and
the tertiary fragmentation pathways in PheSO3, respectively.
Loss of CO2 (44 Da) and C3H5NO2 (87 Da) are observed only for the positively
charged ion. The comparisons of PES for decarboxylation pathway of PheNMe3+ and
PheSO3 do not account the descripencies in fragmentation of these charge-remote Phe.
Even when they both lose some common neutral fragments, the structures of the product
ions are different. Thus, although both PheNMe3+ and PheSO3 are charge-remote Phe,
they behave differently upon collison-induced dissociation.
120
Fig
ure
6.7.
Pro
pose
d F
ragm
enta
tion
Pat
hway
s of
Phe
NM
e 3+
120
121
In order to explain the extensive fragmentation on PheNMe3+, we have
considered the possibility of rearrangement of the amino acid prior to dissociation.
Assuming that the benzene ring and the N,N,N-trimethyl ammonium groups (R) do not
participate in rearrangement, 19 stuctures are possible upon rearrangement of PheNMe3+
(Figure 6.8).
RNH
O
OH
(-13)
NH
O
OH
(-13)
RR
NH
O
O
(-12)
RNH
O
OH
(-5)
R
NH2 O
OH
(-1)
RNH2
O
OH
(0)
RO
O NH2
(0)
RN
OH
H O
(+1)
RN
O
O
(+2)
R NH
RNH OO
O O
(+3) (+3)
R NH O
OH
(+4)
R O
ONH2
(+5)
R
NH2
O
O
(+7)
R
O
ONH2
(+32)
R ON R
O
ONHO H
(+35)(+35)
R N OH
O
(+3)
R O
O
NH
(+7)
Ia Ib Ic Id
IIa IIb IIc IId
IIIa IIIb IIIc IIId
IIIe IIIf IIIg IIIh
IVa IVb IVc
R =
N+
Figure 6.8. Possible rearrangement products of PheNMe3+ and their relative energies in kcal/mol calculated at the B3LYP/6-31+G* level of theory .
122
Unfortunately, most of the structures in Figure 6.8 are too synthetically
challenging to examine the authentic ions. One authentic ion that has been investigated is
the carbamic acid isomer,Ia, which was formed in situ by CID of the boc-protected
phenylethylamine (eq 6.1).
The base peak at m/z 178 observed on CID spectrum of Ia, Figure 6.9, is due to
decarboxylation and this product ion is confirmed to be 2 by using CID (eq 6.2). The
results confirm that amino acid isomers could account for products observed upon CID of
PheNMe3+ .
123
Figure 6.9: CID spectrum of Ia with m/z 223
We propose that rearrangement of PheNMe3+ to Ia is a stepwise process, which
occurs in two steps as shown in eq 6.3. The first process involves a proton shift from an
amine to carbonyl oxygen and the attack of the electron-rich amine to electron-deficient
carbonyl to form a three-membered ring. The second process involves breaking a C-C
bond, a proton transfer from the hydroxyl group to the α-carbon and the regeneration of
carbonyl.
Although a rearrangement similar to that in eq 6.3 is possible for PheSO3, it
appparently does not occur, indicatig that the rearrangement barrier is prohibitive for
negative charged ion.
m/z
120 130 140 150 160 170 180 190 200 210 220 230
Rel
Int
0.0
0.2
0.4
0.6
0.8
1.0
223
179
124
6.5 References
(1) Albrecht, G.; Corey, R. J. Am. Chem. Soc. 1939, 61, 1087.
(2) Marsh, R. Acta Crystallogr. 1958, 11, 654.
(3) Levy, H.; Corey, R. J. Am. Chem. Soc. 1941, 63, 2095.
(4) Donohue, J. J. Am. Chem. Soc. 1950, 72, 949.
(5) Gaffney, J.; Pierce, R.; Friedman, L. J. Am. Chem. Soc. 1977, 99, 4293.
(6) Cohn, E.; McMeekin, T.; Edsall, J.; Blanchard, M. J. Am. Chem. Soc. 1934, 56, 784.
(7) Ellzy, M.; Jensen, J.; Hameka, H.; Kay, J. Spectrochim. Acta, Part A 2003, 59, 2619.
(8) Jensen, J.; MS, G. J. Am. Chem. Soc. 1991, 113, 7917.
(9) Jensen, J.; Gordon, M. J. Am. Chem. Soc. 1995, 117, 8159.
(10) Banerjee, S.; Mazumdar, S. Int. J. Anal. Chem. 2012.
(11) Piraud, M.; Vianey-Saban, C.; Petritis, K.; Elfakir, C.; Steghens, J.; Morla, A.; Bouchu, D. Rapid Commun. Mass Spectrom. 2003, 17, 1297.
(12) El Aribi, H.; Orlova, G.; Hopkinson, A.; Siu, K. J. Phys. Chem. A 2004, 108, 3844.
(13) Koirala, D.; Kodithuwakkuge, S.; Wenthold, P. J. Phys. Org. Chem. 2015, 28, 635.
(14) Cheng, C.; Gross, M. Mass Spectrom. Rev. 2000, 19, 398.
(15) Adams, J. Mass Spectrom. Rev. 1990, 9, 141.
(16) Chuchani, G.; Dominguez, R.; Rotinov, A. Int. J. Chem. Kinet. 1991, 23, 779.
(17) Chuchani, G.; Dominguez, R. Int. J. Chem. Kinet. 1995, 27, 85.
(18) Domingo, L.; Picher, M.; Andres, J.; Safont, V.; Chuchani, G. Chem. Phys. Lett. 1997, 274, 422.
(19) Chuchani, G.; Dominguez, R. Int. J. Chem. Kinet. 1999, 31, 725.
125
(20) Domingo, L.; Picher, M.; Safont, V.; Andres, J.; Chuchani, G. J. Phys. Chem. A 1999, 103, 3935.
(21) Wang, S.; Liu, B.; Sun, K.; Su, Q. J. Chromatogr. A 2004, 1025, 255.
(22) Patterso.JM; Haider, N.; Papadopo.EP; Smith, W. J. Org. Chem. 1973, 38, 663.
(23) Li, J.; Liu, Y.; Shi, J.; Wang, Z.; Hu, L.; Yang, X.; Wang, C. Thermochim. Acta 2008, 467, 20.
126
CHAPTER SEVEN: MOBILE C-H PROTONS IN A PROTON DEFICIENT PEPTIDE
7.1Introduction The significance of amino acids and peptides to the world around us, and to life
itself, cannot be overstated, and, therefore, it is not surprising that their structures,
properties, and reactivity are exceptionally well-characterized, largely due to the
capabilities of mass spectrometry and electrospray ionization.1 The ability for rapid,
accurate analysis of proteins and peptides by mass spectrometry has had a major impact
on the field of proteomics, and allows for the study of the structures and functions of
large proteins in complex biological system.
One of the most important features of using mass spectrometry to identify
proteins and peptides in proteomics is the ability to use tandem mass spectrometry (MSn)
to differentiate isobaric ions and for sequence determination. Therefore, whereas mass
spectral libraries can be used to identify known proteins, amino acid sequences in
unknown proteins can be determined by analysis of ionic fragments. In particular,
fragments observed in peptide dissociation, especially b- and y-type sequence ions, 2
resulting from fragmentation of amide bonds, can be used to determine the sequence from
the C- and N-terminus.2,3
Products obtained upon dissociation of protonated peptides have generally been
explained in terms of the “mobile proton theory”.2,4-6 The model assumes that protons,
127
initially localized on the most basic sites (N-terminus and the side chains of basic amino
acid residues) of protonated peptides, can be transferred to the less basic of sites upon
activation including the various peptide linkages, thereby producing a heterogeneous
population of protonated peptides.2,4,5 The mobile proton model provides a quantitative
framework such that if the peptide sequence and number of protons are known, one can
predict the general appearance of a fragmentation spectrum.4 Although the mobile proton
model is not intended to model the full peptide fragmentation spectrum quantitatively, it
successfully accounts for common dissociation, including aforementioned b- and y-type
ions.
Because peptide b ions are common, stable, species in peptide fragmentation,7 a
better understanding of how they are formed can improve peptide sequencing algorithms
and the current models for peptide fragmentation.2 Consequently several theoretical and
experimental studies have been carried in recent years to better understand the structures
of b ions and the mechanism of their formation.7-27 In this work, we use dipeptides to
investigate the formation of b2 ions.
Many structures have been proposed for b2 ions.4 Initially, an acylium ion was
thought to be the major b2+ ion but it was later discovered to be thermodynamically less
stable than its isomeric cyclic structures.26 Of the structures proposed for b2 ions, the
most common are protonated diketopiperazines and protonated oxazolones.3,13,18,22,27-39
Six-membered cyclic diketopiperazine is formed when the N-terminal amino group act as
a nucleophile and attack at the carbonyl carbon of second amino acid residue. However,
five-membered cyclic oxazolone is formed when the N-terminal carbonyl oxygen act as a
nucleophile and attacks at the carbonyl carbon of second amino acid residue. Which ion
128
forms is mainly determined by the structure of the peptide: A cis conformation of the first
amide bond favors diketopiperazine because it facilitates the nucleophilic attack by
amino group of N-terminal amino acid, Figure 7.1.32,35,39-41 Similarly, trans conformation
of the first amide bond favors oxazolone because it facilitates the nucleophilic attack by
carbonyl oxygen of N-terminal amino acid, Figure 7.1. Although the diketopiperazine is
lower in energy, oxazolone structures result for trans amide because the barrier for the
formation is lower than the barrier for cis-trans isomerization.
COOX
N R2H2N
O
R1
R2NH
COOX
H
OR1
NH2
cis Amide Bond
R2
NH
O
HN
OR1
R2N
O
H2NO
R1
-HXO
-HXO
Oxazolone b2 ion
Diketopiperazine b2 ion
1st residue 2nd residue
1st residue 2nd residue
trans Amide Bond
Figure 7.1. Formation of diketopiperazine from dipeptide with cis amide bond (top) and formation of oxazolone from dipeptide with trans amide bond (bottom)
The nature of the charge on peptides also plays an important role in the
dissociation mechanism. Mass spectrometry studies of peptide and peptide’s dissociation
usually involve the use of protonated42 or metallated 3,43 peptides, often multiply charged
depending on the number of basic sites. Therefore, the excess positive charge allows for
facile intramolecular proton transfer that does not require, inter alia, formation of salt-
129
bridges or other forms of charge separation. In a recent study, we reported the formation
and dissociation of sulfonated phenylalanine, PheSO3-, an anionic amino acid where the
charge is isolated from the α-amino acid moiety by a phenyl spacer. The ion dissociates
only by loss of NH3 to make a product assigned to be an α-lactone (eq 7.1). The proposed
mechanism involves proton transfer within the canonical amino acid to form a
zwitterionic intermediate, which dissociates by intramolecular substitution to make the
lactone (eq 7.1), and therefore nominally illustrates the potential of mobile electron
within anionic systems as well. However, an important feature of all these reactions is
that although energetically unfavorable proton transfer can occur in these reactions upon
activation, it is still generally limited to heteroatom positions, such as amines, amide
nitrogen or carboxyl groups.
In this work, we have used mass spectrometry to examine the dissociation of
dipeptides that include PheSO3¯, namely Gly-Phe*OH and Phe*-GlyOH, where Phe*
refers to PheSO3¯. Unlike protonated and metallated ions, these dipeptides are anionic
and hence proton deficient. However, they still have a canonical dipeptide moiety. In this
130
respect they are similar to protonated dipeptides containing His 29,38-40,44,45 or Arg,
13,24,25,46 which contain basic side chains that sequester positive charge.
We find that the sulfonated dipeptides dissociates similar to what has been
reported for Arg-Gly but the presence of anionic substrate must dissociate that involve
mobile C-H protons due to less number of mobile proton. We find Gly-Phe*OH and
Phe*-GlyOH both dissociates to form the same b2 ions after rearranging to a common
structure of the canonical amino acid.
7.2 Experimental
Experiments were carried out using a commercial LCQ-DECA (Thermo Electron
Corporation, San Jose, CA) quadrupole ion trap mass spectrometer, equipped with
electrospray ionization (ESI) source. Samples were dissolved in methanol: water (1:1)
and introduced into the source at a flow rate of 10 µl/min. Electrospray and ion focusing
conditions were varied so to maximize the signal of the ion of interest. Dissociation of
ions was carried out by using MSn experiments with mass-selected ions in the cell, with
the helium buffer serving as the collision target. Reactant ions for CID were isolated at
qz = 0.250 and with a mass-width sufficient to avoid off-resonance excitation of the
131
mass-selected ions. The energy of collision-induced dissociation (CID) in the cell is
reflected in the “normalized collision energy,” which ranges from 0 – 100%.
7.3 Synthesis
7.3.1 Synthesis of Amino Acid Esters
A 50 mg sample of the amino acid was dissolved in 10 ml of alcohol and cooled
to 0 C. Esterification was initiation by adding 3 ml of SOCl2, dropwise, into the cold
amino acid solution. After the solution warmed to room temperature, it was refluxed
overnight to get desired amino acid ester.
7.3.2 Synthesis of Gly-Phe*OH and Gly-Phe*OMe Dipeptides
0.09 mmol of Boc-GlyOH was dissolved in 15 ml of dichloromethane (DCM) and
0.09 mmol of coupling reagent (PyBop) was added. The solution was cooled down to
0 °C and 0.14 mmol of base (DIEA) was then added dropwise. After stirring for 20 min,
the solution was brought to a room temperature then 0.09 mmol of Phe*OH or
Phe*OMe was added and stirred for additional 40 min. Excess DCM was evaporated in
vacuum and formation of desired product was confirmed by mass spectrometry. The use
of N-Boc glycine prevents the coupling reaction in reverse order of amino acids for the
formation of Xxx-Gly dipeptide since the N is protected, where Xxx is amino acid, where
Xxx is Phe*OH in this case. Hence, the peak observed at desired m/z is Gly-Xxx and
none of Xxx-Gly. The Boc group was then removed by dissolving product in excess
trifluoroacetic acid (TFA) for 20 min and evaporating TFA in vacuum. In addition,
132
compound ii can be hydrolyzed to corresponding acid by treating their aqueous solution
with concentrated sulfuric acid.
7.3.3 Synthesis of Phe*-GlyOH and Phe*-GlyOMe Dipeptides
We used similar procedure in this section as explained above for Gly-Phe*OH
with some modifications. 0.09 mmole of Gly-OMe and 0.09 mmole of Phe*OH were
dissolved in 15 ml of DCM and 0.09 mmol of PyBop was added. The solution was cooled
down to 0°C and 0.14 mmol of DIEA was then added dropwise. The solution was stirred
at 0°C for 20 min and at room temperature for additional 40 min. Unlike reaction 1, we
did this reaction in single step because we want to reduce the self-coupling of Phe*OH to
form Phe*-Phe*OH dipeptide. This self-coupling reaction could be completely stopped
by using Boc-Phe*OH but Boc-Phe*OH is not commercially available and we did not
have any success on synthesizing it. To obtain the desired dipeptide, we used unmodified
Phe*OH with glycine ester. The benefit of using Gly-OMe is that it prevents Gly-Xxx
formation, which makes mass spectrum unambiguous. In addition, Phe*-GlyOMe can be
hydrolyzed to Phe*-GlyOH by treating its aqueous solution with concentrated sulfuric
acid.
7.3.4 Synthesis of Diketopiperazine
Synthesis of diketopiperazine (I) has been explained previously.47,48 Briefly, the
solution containing 50 mg of Boc-Gly-Phe*OMe and 10 ml formic acid (98%) was
stirred at room temperature for 2 hr. Excess formic acid was removed in vaccuo and the
crude product was dissolved in 15 ml of sec-butyl alcohol and 5 ml of toluene. The
133
solution was boiled for 3 hr and the solvent level maintained by addition of fresh butanol.
After concentrating the solution to 5-10 ml and cooling to 0C the products were filtered
off and studied on mass spectrometry.
7.3.5 Synthesis of Gly-Phe* Oxazolone
Synthesis of oxazolone has been explained previously. 48 Briefly, the solution
containing 10 umol of Boc-Gly-Phe*OH and 15 umol of 1, 3,-Dicyclohexylcarbodiimide
(DCC) were dissolved in 10 ml of dichloromethane. The solution was stirred at room
temperature for 3 hr. Excess solvent was removed in vaccuo and the resulting crude
product was dissolved in 3 ml TFA to produce Gly-Phe*-oxazolone (II).
134
7.4 Results and Discussion
7.4.1 Analysis of Gly-Phe*OH and Phe*-GlyOH
The CID (MS2) mass spectra of Gly-Phe*OH and Phe*-GlyOH (m/z 301) are
shown in Figure 7.2. Both isomers give the same products with the same intensities.
Primary product ions include m/z 284 (M-NH3), m/z 283(M-H2O), m/z 257(M-CO2), and
m/z 244 (M-C2ONH3, net loss of glycine). The fact that both Gly-Phe*OH and Phe*-
GlyOH give the same spectra indicates that they rearrange to a common structure before
dissociation. Rearrangement of dipeptides upon low-energy CID has been reported
previously by O’Hair and co-workers for protonated Arg-Gly/Gly-Arg.25 As described in
the introduction, protonated Arg-Gly/Gly-Arg is similar to Phe*-GlyOH/Gly-Phe*OH
because it is a canonical dipeptide with the charge on the side chain. Therefore, the
rearrangement of Gly-Phe*OH and Phe*-GlyOH dipeptides is likely similar to what
was proposed previous by O’Hair and co-workers for protonated Arg-Gly and Gly-Arg
dipeptides, involving proton transfer within the dipeptide to form zwitterionic structures,
which can rearrange to cyclic structure and eventually rearrange to form the common
acid anhydride intermediate (Figure 7.3). Consequently, any of the structures in Figure
7.3 can be the common intermediate leading to the dissociation product(s).
135
Figure 7.2. Mass Spectra: a) CID of Gly-Phe*OH and b) CID of Phe-Gly*OH
136
Figure 7.3. Proposed mechanism for formation of common structure from Phe*-GlyOH and Gly-Phe*OH
7.4.2 Analysis of Gly-Phe*OMe and Phe*-GlyOMe:
Support for the mechanism shown in Figure 7.3 comes from the experiments
involving methyl esters. O’Hair and co-workers25 have shown previously that
methylation of the C-terminus carboxylic acid inhibits the rearrangement reaction (Figure
7.3) of protonated Arg-Gly and Gly-Arg, presumably by blocking the initial proton
transfer step. Similarly, we have found that esterification of Gly-Phe*OH and Phe*-
137
GlyOH inhibits their rearrangement as well. The CID spectra of Gly-Phe*OMe and
Phe*-GlyOMe, shown in Figure 7.4, are significantly different in both the identities of
the products and the intensities. Although both ions dissociate to form products m/z 283
(M-MeOH), m/z 266 (M-MeOH-NH3) and m/z 170, the relative intensities are very
different. In particular, whereas the b2 ion (M-CH3OH @ m/z 283) is one of the dominate
peaks upon CID of Phe*-GlyOMe, it is very weak upon CID of Gly-Phe*OMe. In
addition, peaks at m/z 298, m/z 240, m/z 226, m/z 198, m/z 185 and m/z 171 are observed
solely on CID of Phe*-GlyOMe spectra, whereas m/z 266, m/z 241, and m/z 197 are
observed solely on CID of Gly-Phe*OMe spectra.
138
Figure 7.4. Mass Spectra: a) CID of Gly-Phe*OMe and b) CID of Phe*-GlyOMe
The M-CH3OH ions observed upon CID of Gly-Phe*OMe and Phe*-GlyOMe
are the b2 ions. The fact that b2 ions can be observed with the dipeptides (Gly-Phe*OH
and Phe*-GlyOH) and the methyl esters illustrates why they are significant in standard
peptide sequencing: they are not dependent on the substitution at the carboxyl end. The
MS3 spectra of b2 ions obtained from Gly-Phe*OMe and Phe*-GlyOMe shown in
Figure 7.5a and 7.5b, are also different, consistent with different ions giving different
139
products. The CID spectra of the b2 ions obtained from Phe*-GlyOH and Gly-Phe*OH
agree with that from that obtained from Phe*-GlyOMe.
The CID spectra of the b2 ions can be compared to those for authentic ions. Figure
7.5c shows the CID spectrum of 1, whereas Figure 7.5d is for that of 2. There is a
marginal similarity between CID of the b2 ions obtained from Gly-Phe*OMe (Figure
7.5a) and for the CID 1 (Figure 7.5c). Peaks in common include m/z 255 and m/z 170.
However, the relative intensities do not match, and there are peaks in the CID of b2 ion
that are not found for CID 1. Therefore, although the CID spectrum is partially consistent
with 1, not all of the b2 ions can have that structure and there would need to be an
isobaric mixture.
140
Figure 7.5. Mass Spectra: CID of b2 ions a) MS3 spectra of Gly-Phe*OMe and b) MS3 spectra of Phe*-GlyOMe c) CID of 1 d) CID of 2
141
The fact that Phe*-GlyOMe, which has no acidic proton, forms same b2 ion as
that of Phe*-GlyOH and Gly-Phe*OH indicates that the formation of b2 ions does not
require the rearrangement of precursor ions to a common structure, as shown in Figure
7.2. In addition, the CID spectrum of this b2 ion does not match the CID spectra of 1 or
that of 2. We have used isotopic labelling experiment to investigate further the formation
and structure of b2 ion from Phe*-GlyOMe.
The oxazolone structures for the b2 ions that would be obtained from Gly-
Phe*OMe and Phe*-GlyOMe are 2 and 3 respectively. An important difference between
the two structures involves the origin of the carbonyl in the oxazolone structure, which
originates from the C-terminus. Therefore, the carbonyl in 2 would come from the Phe*
residue, whereas that in 3 comes from the Gly residue. If the b2 ion obtained from Phe*-
GlyOMe has an oxazolone structure, then CID process that involves loss of the carbonyl
should be losing the glycine carbonyl. To test for this possibility, we examined Phe*-
GlyOMe with glycine labelled with 13C at the carbonyl (Figure 7.6). Upon CID, the CO
and CO2 losses both contain the 13C label and loss of non-labelled CO and CO2 is not
obtained. These results are consistent with the structure of 3 for the b2 ion.
142
Figure 7.6. Mass Spectrum: CID of Phe*-Gly (1-13C) OMe
7.4.3 Deuterium Labelled Phe*-GlyOMe
We have also used deuterated labelling to investigate the mechanism of formation
of the b2 ions. Because the b2 ion formed upon dissociation of Phe*-GlyOMe is same as
that from Phe*-GlyOH/Gly-Phe*OH, we have focused on CID of Phe*-GlyOMe
because it does not undergo the rearrangement shown in Figure 7.3.
In order to determine which proton is involved in methanol loss, we examined
three deuterated labelled compounds d3-Phe*-GlyOMe, Phe*-Gly (2, 2-d2) OMe, and
d5- Phe*-GlyOMe. H/D exchange of labile protons was carried out in 50:50 D2O and
MeOD solution. The CID spectra of these compounds are shown in Figure 7.7. Upon
CID, d3-Phe*-GlyOMe loses MeOD and MeOH in a ratio of about 2:1 (Figure 7.7a).
143
The loss of MeOD is consistent with what is expected for b2 ion formation, 3, with loss of
methoxy from the C-terminus and loss of a mobile proton from one of the nitrogen
position. Loss of CH3OH is surprising, especially as the major product, because it
requires loss of hydrogen from a carbon position, which would not normally be
considered a “mobile” proton. Although the deuterium labelling shows those protons on
carbon are involved in the dissociation, it does not specify which carbons, whether they
are α-carbon on Gly or Phe* or from the aliphatic or aromatic region of the side-chain.
Addition deuterium labelling experiments, however, provide further insight. The
spectrum for Phe*-GlyOMe, where the Gly is labelled with deuterium in the α -positon
(2, 2-d2), is shown in Figure 7.7b. Again, the ion dissociates by loss of MeOH and
MeOD, but in this case, the intensities are reversed, with loss of MeOD being the major
b2 product.
Loss of CH3OD as the major pathway for b2 ion formation confirms that carbon-
based hydrogens are involved, and, in particular, it is the proton on the α-position of
glycine. However, it does not rule out participation of other protons, such as that at the a-
position of Phe* or those on the side chain. To test for the possibility, we also deuterated
the nitrogen positions. The CID spectrum of the d5-Phe*-GlyOMe is shown in Figure
7.7c. In this case, the b2 ion is formed almost exclusively (>98%) by loss of CH3OD.
Therefore, although b2 ion can be formed by loss of proton from nitrogen, the majority is
formed by loss of proton from the α-position of glycine.
A mechanism that occurs for the observed products is shown in Figure 7.8.
Nucleophilic attack of the Phe* carbonyl oxygen at the Gly carbonyl carbon
accompanied by proton transfer leads to the hemiacetal-like structure, X. Loss of alcohol,
144
with proton loss from the OH in X, leads to the standard oxazolone, accounting for loss
of the proton from N. For this pathway, the carbonyl in the oxazolone originates from the
Gly, consistent with the 13C labelling experiments. Alternatively, loss of alcohol with the
proton from the ring forms the hydroxyl-oxazole, the enol structure of the oxazolone.
145
Figure 7.7. Mass Spectra: CID of deuterated sample a) d3-Phe*-GlyOMe b) Phe*-Gly (2, 2-d2) OMe c) d5- Phe*-Gly (2, 2-d2) OMe
146
Figure 7.8. Proposed fragmentation pathway of Phe*-GlyOR’
7.4.4 Computational Results
Unlike phenols, hydroxyoxazoles are typically not more stable than the
corresponding oxazolones. At the MP2/6-31+G*//B3LYP/6-31+G* level of theory, the
simple oxazolone is computed to be 15.4 kcal/mol lower in energy than the
147
hydroxyoxazole, Table 7.1. However, interaction between the hydroxy group and the
sulfonate charge in the b2 is predicted to stabilize the enol structure.
The relative energies of isomeric b2 ion structures are shown in Table 7.1. The
most stable product is the diketopiperazine (1), and the 2 and 3 oxazolone b2 ions are
calculated to be 14 and 20 kcal/mol higher in energy, respectively. The relative energies
of the diketopiperizine and oxazolones are consistent with what has been found in
previous studies. However, at the MP2/6-31+G* level of theory, the lowest energy
structure, aside from the diketopiperizine, is predicted to be the hydroxyoxazole obtained
from Phe*-GlyOH, Figure 7.9. In contrast, the hydroxyoxazole obtained from Gly-
Phe*OH is significantly higher in energy. The stability of the enol structure for the
Phe*-GlyOH b2 can be attributed to a favorable hydrogen bond interaction between the
OH and the sulfonate group, as shown in Figure 7.9, and accounts for the preference for
losing proton from carbon, as opposed to from nitrogen. The oxazolone structure
obtained from Gly-Phe* also has a hydrogen bond, between the N-H and sulfonate,
which accounts for its stability. The other structures are not capable of having
interactions with the sulfonate. The optimized geometries are shown in Figure 7.9.
Table 7.1. Calculated Relative Energies of b2 Ionsa
Ion MP2/6-31+G* Diketopiperizine 1 0.0 Phe*-Gly Oxazolone 3 20 Phe*-Gly Oxazolone enol 4 12 Gly-Phe* Oxazolone 2 14 Gly-Phe* Oxazolone enol 5 26
aRelative electronic energies (not corrected) in kcal/mol at B3LYP/6-31+G* optimized geometries
148
Figure 7.9: Lowest energy structures of 1, 3, 4, 2, and 5 respectively
7.5 Conclusion
The charge-remote fragmentation of dipeptide is reported. We discovered that
Phe*-GlyOH and Gly-Phe*OH rearranges to common structure before dissociation. The
rearrangement is blocked by methylation. The b2 ion formed from dissociation of Phe*-
GlyOMe is same as that from Phe*-GlyOH and Gly-Phe*OH, which provided further
inside on mechanism of dissociation since first step does not need to be proton transfer.
Combination of several experimental and theoretical results suggest Phe*-Gly oxazolone
and Phe*-Gly oxazolone-enol are predominate structures of the b2 ions for the model
compound we studied. Most importantly, we have shown that C-H protons are involved
in the dissociation, effectively behaving as “mobile protons” in the proton-deficient
peptide.
149
7.6 References
(1) Banerjee, S.; Mazumdar, S. Int. J. Anal. Chem. 2012.
(2) Wysocki, V.; Tsaprailis, G.; Smith, L.; Breci, L. J. Mass Spectrom. 2000, 35, 1399.
(3) Lee, V.; Li, H.; Lau, T.; Siu, K. J. Am. Chem. Soc. 1998, 120, 7302.
(4) Wysocki; Cheng; Zhang; Hermann; Beardsley; Hilderbrand. In Principles of Mass spectrometry Applied to Biomolecules 2006.
(5) Boyd, R.; Somogyi, A. J. Am. Soc. Mass. Spectrom. 2010, 21, 1275.
(6) Dongre, A.; Jones, J.; Somogyi, A.; Wysocki, V. J. Am. Chem. Soc. 1996, 118, 8365.
(7) Yalcin, T.; Khouw, C.; Csizmadia, I.; Peterson, M.; Harrison, A. J. Am. Soc. Mass. Spectrom. 1995, 6, 1165.
(8) Rodriquez, C.; Shoeib, T.; Chu, I.; Siu, K.; Hopkinson, A. J. Phys. Chem. A 2000, 104, 5335.
(9) Harrison, A. Mass Spectrom. Rev. 2009, 28, 640.
(10) Ambihapathy, K.; Yalcin, T.; Leung, H.; Harrison, A. J. Mass Spectrom. 1997, 32, 209.
(11) Bythell, B.; Knapp-Mohammady, M.; Paizs, B.; Harrison, A. J. Am. Soc. Mass. Spectrom. 2010, 21, 1352.
(12) Bowie, J.; Brinkworth, C.; Dua, S. Mass Spectrom. Rev. 2002, 21, 87.
(13) Zou, S.; Oomens, J.; Polfer, N. Int. J. Mass spectrom. 2012, 316, 12.
(14) Chen, X.; Tirado, M.; Steill, J.; Oomens, J.; Polfer, N. J. Mass Spectrom. 2011, 46, 1011.
(15) Chen, X.; Yu, L.; Steill, J.; Oomens, J.; Polfer, N. J. Am. Chem. Soc. 2009, 131, 18272.
(16) Chu, I.; Shoeib, T.; Guo, X.; Rodriquez, C.; Lan, T.; Hopkinson, A.; Siu, K. J. Am. Soc. Mass. Spectrom. 2001, 12, 163.
(17) Bythell, B.; Csonka, I.; Suhai, S.; Barofsky, D.; Paizs, B. J. Phys. Chem. B 2010, 114, 15092.
150
(18) Farrugia, J.; O'Hair, R.; Reid, G. Int. J. Mass spectrom. 2001, 210, 71.
(19) Grewal, R.; El Aribi, H.; Harrison, A.; Siu, K.; Hopkinson, A. J. Phys. Chem. B 2004, 108, 4899.
(20) Harrison, A. J. Am. Soc. Mass. Spectrom. 2009, 20, 2248.
(21) Knapp-Mohammady, M.; Young, A.; Paizs, B.; Harrison, A. J. Am. Soc. Mass. Spectrom. 2009, 20, 2135.
(22) Yoon, S.; Chamot-Rooke, J.; Perkins, B.; Hilderbrand, A.; Poutsma, J.; Wysocki, V. J. Am. Chem. Soc. 2008, 130, 17644.
(23) Wysocki, V.; Resing, K.; Zhang, Q.; Cheng, G. Methods 2005, 35, 211.
(24) Forbes, M.; Jockusch, R.; Young, A.; Harrison, A. J. Am. Soc. Mass. Spectrom. 2007, 18, 1959.
(25) Farrugia, J.; O'Hair, R. Int. J. Mass spectrom. 2003, 222, 229.
(26) Nold, M.; Wesdemiotis, C.; Yalcin, T.; Harrison, A. Int. J. Mass Spectrom. Ion Processes 1997, 164, 137.
(27) Wang, D.; Gulyuz, K.; Stedwell, C.; Polfer, N. J. Am. Soc. Mass. Spectrom. 2011, 22, 1197.
(28) Polce, M.; Ren, D.; Wesdemiotis, C. J. Mass Spectrom. 2000, 35, 1391.
(29) Perkins, B.; Chamot-Rooke, J.; Yoon, S.; Gucinski, A.; Somogyi, A.; Wysocki, V. J. Am. Chem. Soc. 2009, 131, 17528.
(30) Paizs, B.; Suhai, S. J. Am. Soc. Mass. Spectrom. 2004, 15, 103.
(31) Paizs, B.; Lendvay, G.; Vekey, K.; Suhai, S. Rapid Commun. Mass Spectrom. 1999, 13, 525.
(32) Morrison, L.; Chamot-Rooke, J.; Wysocki, V. Analyst 2014, 139, 2137.
(33) Eckart, K.; Holthausen, M.; Koch, W.; Spiess, J. J. Am. Soc. Mass. Spectrom 1998, 9, 1002.
(34) Bythell, B.; Somogyi, A.; Paizs, B. J. Am. Soc. Mass. Spectrom. 2009, 20, 618.
(35) Bythell, B.; Suhai, S.; Somogyi, A.; Paizs, B. J. Am. Chem. Soc. 2009, 131, 14057.
151
(36) Sinha, R.; Erlekam, U.; Bythell, B.; Paizs, B.; Maitre, P. J. Am. Soc. Mass. Spectrom. 2011, 22, 1645.
(37) Paizs, B.; Suhai, S. J. Am. Soc. Mass. Spectrom. 2003, 14, 1454.
(38) Farrugia, J.; Taverner, T.; O'Hair, R. Int. J. Mass spectrom. 2001, 209, 99.
(39) Armentrout, P.; Clark, A. Int. J. Mass spectrum. 2012, 316, 182.
(40) Gucinski, A.; Chamot-Rooke, J.; Nicol, E.; Somogyi, A.; Wysocki, V. J. Phys. Chem. A 2012, 116, 4296.
(41) Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2001, 15, 2307.
(42) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508.
(43) Dunbar, R.; Polfer, N.; Berden, G.; Oomens, J. Int. J. Mass spectrum. 2012, 330, 71.
(44) Dunbar, R.; Steill, J.; Polfer, N.; Oomens, J. Int. J. Mass spectrum. 2009, 283, 77.
(45) Tsaprailis, G.; Nair, H.; Zhong, W.; Kuppannan, K.; Futrell, J.; Wysocki, V. Anal. Chem. 2004, 76, 2083.
(46) Tsaprailis, G.; Somogyi, A.; Nikolaev, E.; Wysocki, V. Int. J. Mass spectrum. 2000, 195, 467.
(47) Nitecki, D.; Halpern, B.; Westley, J. J. Org. Chem. 1968, 33, 864.
(48) Uraguchi, D.; Asai, Y.; Ooi, T. Angew. Chem. Int. Ed. 2009, 48, 733.
VITA
152
VITA Damodar Koirala was born in November 1985 to Bishnu Prasad and Radhika
Koirala in Pokhara Nepal. He is youngest three children, and is loved, cared, and
supported by bother Kamal Koirala and sister Kamala Bhattarai throughout his career.
Upon graduating from high school from VS Niketan College, Kathmandu, he joined
University of Wisconsin-Superior for undergraduate degree in Aug 2005. He started with
Biology major but later switched to Chemistry and Mathematic majors. He graduated
with Bachelor of Science degree on Dec 2010. He worked for a year at UW-Superior
after graduation and joined Purdue University to pursue his PhD degree in Chemistry on
Aug 2011. He joined Dr. Paul Weldhold’s lab on Nov 2011 and soon started expertizing
on application of mass spectrometer.
On May 2013, Damodar got married to his beautiful wife Binita K. Koirala. They
then had a baby girl, Erina Koirala, on April of 2014. Both, Binita and Erina, supported
him in all expects since they arrived to his life. Currently, they live in Lafayette, IN, with
Damodar’s brother Kamal, sister-in-law Goma, 3 years old nephew Kritan and 2 and half
months old niece Keva Koirala. They are a big happy family.
Damodar will graduate with a Ph.D. in Chemistry in August of 2016. Eventually,
he desires to become an industrial chemist or a Chemistry teacher in a small
undergraduate University.
PUBLICATIONS
Mass spectrometric study of the decompositionpathways of canonical amino acids andα-lactones in the gas phaseDamodar Koiralaa, Sampath Ranasinghe Kodithuwakkugea‡ andPaul G. Wentholda*
The dissociation pathways of a gas-phase amino acid with a canonical (non-zwitterionic) α-amino acid moiety are studied byusing mass spectrometry. Investigation of the canonical amino acid moiety is possible because the ionized amino acid, asulfonated phenylalanine, has a charge center that is separated from the amino acid, and dissociation occurs by charge-remote fragmentation. The amino acid is found to dissociate only by loss of NH3 upon collision-induced dissociation to form asubstituted α-lactone. The dissociation is consistent with what has been observed previously upon pyrolysis of other α-substitutedcarboxylic acids. Decarboxylation, which has also been reported previously for amino acid pyrolysis, is not observed, likely becausethe product would be a high-energy, ammonium ylide. The resulting α-lactone is found to undergo dissociation by decarbonylationto give an aldehyde, and by loss of CO2. Decarboxylation is calculated to occur through a transition state involving hydride shiftcoupled with lactone ring-opening. The transition state is found to be stabilized by the negative charge, and therefore, decar-boxylation is more favorable for anions. The results show that remote ionic groups can be used as mostly inert charge carriersto enable mass spectrometry to be used to investigate the gas-phase physical and chemical properties of different types offunctional groups, including amino acids. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: amino acid dissociation; gas-phase amino acids; mass spectrometry
INTRODUCTION
The significance of amino acids to the world around us, and tolife itself, cannot be overstated, and, therefore, it is not surprisingthat their structures, properties, and reactivity are exceptionallywell-characterized, at least in solution. However, owing to theirgeneral lack of volatility, much less is known about the structureand reactivity of amino acids in the gaseous state. Gaseousamino acids are rare, but would be highly significant if reportsof interstellar glycine,[1,2] for example, can be confirmed. In amore practical sense, the gas phase serves as a reasonable ap-proximation for a low dielectric, hydrophobic environment, andso investigation of the gas-phase (solvent free) properties ofamino acids provides insight into the structures of amino acidsin lipid membranes.[3] Finally, regardless of any biological signif-icance, results of studies of gaseous amino acids can be com-pared with those for substituted carboxylic acids to determine,inter alia, the effect of the amino group on the gas-phase struc-ture and reactivity.One of the most important differences between gaseous and
condensed phase amino acids is that, because charge separationis unfavorable in the gas phase, the solvent-free molecules willnot have zwitterionic structures, Z, like those in the condensedphase, but will have non-ionic, canonical (C) structures instead.[4–7]
In fact, zwitterionic structures of gas-phase amino acids are so un-expected that many mass
spectrometric studies have been carried out to try to elucidatethose factors, such as solvation or ion pairing that can lead tostable ionic structures in various amino acids.[8]
Recent advances in spectroscopic methods in the last decadehave provided more detailed insight into the gas-phase struc-tures, including details of the modes of internal hydrogen bond-ing.[9–11] The challenges in these experiments are twofold: on theone hand, because the spectroscopy experiments generally re-quire a good chromophore for ultraviolet absorption, they workbest for amino acids with aromatic side chains. However, thoseamino acids are among the less volatile, and therefore, creatinggaseous samples is difficult. Smaller, aliphatic amino acids aremore volatile, but lack a good chromophore to facilitateresonance-2-photon absorption, which is generally required forthese types of experiments. It is a testament to modern spectro-scopic technology that we do have spectra for both aromaticand aliphatic amino acids.[12–15] Gas-phase photoionization of al-iphatic amino acids has also been reported.[16]
Amino acids have been extensively studied using mass spec-trometry. However, in these studies, the amino acids are typicallyionized in some way, such as via protonation or metallation tocreate positively charged ions or by deprotonation to form neg-
* Correspondence to: P. G. Wenthold, Department of Chemistry, Purdue University,560 Oval Drive, West Lafayette, IN 47907, USA.E-mail [email protected]
‡ Deceased, 2012
a D. Koirala, S. R. Kodithuwakkuge, P. G. WentholdDepartment of Chemistry, Purdue University, 560 Oval Drive, West Lafayette,IN, 47906, USA
Research Article
Received: 14 January 2015, Revised: 05 May 2015, Accepted: 11 May 2015, Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/poc.3464
J. Phys. Org. Chem. 2015 Copyright © 2015 John Wiley & Sons, Ltd.
153
atively charged ions. Consequently, the properties of ionizedamino acids investigated by usingmass spectrometry are generallynot comparable with those of the “neutral” (zwitterionic or canon-ical) amino acids. Here, we describe an investigation of canonicalamino acids by using “charge-remote fragmentation,”[17,18] wheredissociation of an ion occurs, as the name suggests, at locationsremote from the charge site. Charge-remote fragmentation is com-monly used as an analytical tool to determine the structures ofbiological molecules. Lipids[17,18] are particularly amenable to thistechnique, as they fragment in the hydrocarbon chains. However,charge-remote fragmentation has also been used to examinestructures of other biologically relevant molecules such as drugsand their metabolites and peptides.[17,18]
Adams and Gross[19] have proposed that charge-remote frag-mentation, where the charge does not participate in the frag-mentation, is analogous to thermolysis of the gas-phase neutralmolecule. If this is true, then it may be possible to use charge-remote fragmentation
electrosprayed ions as an alternative to pyrolysis in order to studythe gaseous decomposition of otherwise non-volatile organicmolecules. In this study, we examined sulfonated phenylalanine,PheSO3
�, a sulfonate anion with a separate canonical amino acidmoiety. The gas-phase reactivity of amino acids is similarly poorlyknown. The lack of volatility of amino acids has made even nomi-nally simple experiments such as gas-phase pyrolysis exceedinglydifficult, and these experiments generally require generation ofthe amino acid in situ by pyrolysis of appropriate volatile precur-sors. For example, Chuchani and co-workers[20] reported that theycould examine the pyrolytic dissociation of amino acids generatedby decomposition of the corresponding ethyl ester derivatives(Eqn 1). By using this approach, they found that N-substitutedglycines, such as N,N-dimethylglycine,[20] N-phenylglycine,[21]
and N-benzylglycine,[22] for example, dissociate by loss of CO2,as shown in Eqn 1, in the gas phase. In contrast, Al-Awadi andco-workers found that gaseous N-phenylalanine does not un-dergo decarboxylation upon pyrolysis, but dissociates into ani-line, CO, and acetaldehyde, as shown in Eqn 2,[23] in a processthat is similar to what is commonly observed for α-substitutedcarboxylic acids (Eqn 3).[24–36]
By using mass spectrometry, we have examined the decomposi-tion of the gaseous amino acid, PheSO3
�, which contains a sulfo-nate charge and a canonical α-amino acid. We show thatPheSO3
� dissociates initially by fragmentation of the amino acidby the pathway shown in Eqn 3 to form the α-lactone, and direct de-carboxylation (Eqn 1) is not observed. The α-lactone product isfound to fragment by decarbonylation, as expected (Eqn 3) but isalso found to undergo decarboxylation to form the styrene product.Electronic structure calculations find that the presence of the sulfo-nate charge has little effect on the energetics of these dissociationpathways, indicating that the results can be fairly interpreted as rep-resentative of what would happen with the non-ionized derivative.
EXPERIMENTAL
Experiments were carried out using a commercial LCQ-DECA(Thermo Electron Corporation, San Jose, CA, USA) quadrupole iontrap mass spectrometer, equipped with electrospray ionization(ESI). Ions were introduced by spraying dilute aqueous solutionsof commercially available substrates, except for the cis-cinnamicacid and 4-(acryloyloxy)benzenesulfonate, which were synthesizedas described in the succeeding text; solution details are providedas Supporting Information. Electrospray and ion focusing condi-tions were varied so to maximize the signal of the ion of interest.Dissociation of ions can be carried out during ion injection by usinghigh injection voltages, or can be carried out by using MSn exper-iments with mass-selected ions in the cell, with the helium bufferserving as the collision target. Reactant ions for CID were isolatedat qz = 0.250 and with a mass width sufficient to avoid off-resonance excitation of the mass-selected ions. The energy ofcollision-induced dissociation (CID) in the cell is reflected in the“normalized collision energy,” which ranges from 0% to 100%.
Synthesis of (Z)-4-(3-ethoxy-3-oxoprop-1-en-1-yl)benzenesulfonate (deprotonated ethyl 4′-sulfo-cis-cinnamate)
A solution of ethyl diphenyl phosphonoacetate (0.16g, 0.50mmol) intetrahydrofuran (30ml)was treatedwith Triton B (0.27ml, 0.60mmol)at �78 °C for 15min. A solution of p-sulfonated benzaldehyde(0.12g, 0.60mmol in 5ml MeOH) was then added dropwise, andthe resulting mixture was stirred at �78 °C for 1h. The reaction wasquenchedwith 5ml of deionizedwater. ESImass spectra of the crudeproduct indicated a mixture of m/z 255 (the sulfonated ethylcinnamate) and m/z 249, which is assigned to a (PhO)2PO2
� by-product. Nuclearmagnetic resonance spectroscopy of the crudemix-ture in D2O contained doublets at 6.0 and 6.98ppm (J=12.4Hz),which could be assigned to the cis-cinnamate based on comparisonwith the previously reported cis-structures. In contrast, the clearlyobserved NMR peak for the authentic trans isomer is a doublet at6.49ppm with J=15.4. From the NMR spectrum of the cis-sample,we estimate a cis/trans ratio of about 98:2, consistent with the selec-tivities obtained previously using this procedure. The ethyl ester wasintroduced into the mass spectrometer by electrospray andconverted to the carboxylic acid by CID, and elimination of ethylene.
Synthesis of 4-(acryloyloxy)benzenesulfonate (PASO3�)
4-(Acryloyloxy)benzenesulfonate was prepared by mixing 1ml of4-hydroxybenzenesulfonic acid sodium salt dihydrate with 3mlof acryloyl chloride (CH2¼CHCOCl). The neat mixture was stirredfor about 15min at 35°, and 1ml of (i-Pr)2Net was added. The so-lution was stirred approximately 15min, after which one drop of
(1)
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the crude mixture was added to 1ml of a 50/50 mixture of waterand methanol and examined by ESI mass spec. The main prod-ucts observed in the mass spectrum of the crude mixture arethe hydroxybenzenesulfonate, a product with m/z 253, which islikely HO3SC6H4SO4
�, and a product at m/z 227, which is thephenyl acrylate.
Computational methods
Amino acid conformations were surveyed by using the MonteCarlo Multiple Minimum search method, with the Merck MolecularForce Field (MMFFs) as implemented in MacroModel (version9.6).[37] Multiple starting conformations were utilized, includingcanonical and zwitterionic structures, to improve the chances offinding the key low-energy structures. All of the unique conforma-tions calculated with the MMFFs force field to be within 100 kJ/molof the lowest energy structure were identified and used as startingpoints for B3LYP/6-31+G* geometry optimizations.Stationary points were confirmed to be minima or saddle
points from the computed vibrational frequencies. Reported en-ergies are electronic energies, computed at the MP2/6-311+G**level of theory at the B3LYP geometries, designated MP2/6-311 +G**//B3LYP/6-31 +G*, unless noted, and are not correctedfor zero-point energies or thermal energy contributions. Thelarger basis set is used to provide a good description of the vander Waals interactions and polarization of charge. Calculationswere carried out using QCHEM version 4.0[38] and Gaussian.[39]
RESULTS
The mass spectrum of an aqueous solution of PheSO3�, shown in
Fig. 1(a), is very clean, with essentially only peaks correspondingto PheSO3
�. Considering the large difference in acidities ofsulfonic and carboxylic acids,[40] it is not surprising that thesulfonate structure, depicted as PheSO3
�, is computed (B3LYP/6-31+G*) to be more than 20 kcal/mol more stable than the carbox-ylate structure, similar to what has been reported previously forsulfated peptides.[41] Although mixtures of ion isomers have beenfound for amino acid anions when the acidities of the differentgroups are similar,[42,43] the large difference in the acidities of thecarboxylic and sulfonic acids should result in an ion that consistsof a sulfonate charge with separate amino acid moiety. Althoughthe extent of interaction between the charge and the reactivegroup is always a question in these studies, the distance to thecharge is too great for any significant interaction to occur. In addi-tion, the saturated carbons that separate the amino acid from thering prevent any conjugative interactions with the charge.Calculations have been carried out to determine the structure of
PheSO3� in the gas phase. A conformational search identified
more than 20 unique low-energy conformations (within50 kJ/mol of minimum) of the ion, and all but one which have ca-nonical structures. Qualitative depictions of the five lowest energycanonical conformations and the lowest energy zwitterionic struc-ture are shown in Fig. 2. Figure 2 also shows the relative energies ofeach stationary point, optimized at the B3LYP/6-31+G* level oftheory. The most stable conformation has the carboxylic acid moi-ety anti to the aromatic ring, with a hydrogen bond between the –OH proton and the amine nitrogen. Indeed, the anti-conformation,when possible, is generally preferred for the structures examinedin this work. The conformation with the –COOH and aromatic ringgauche is higher in energy by about 2.5 kcal/mol. Alternate hydro-gen bonding arrangements, such as NH– or OH to carbonyl
oxygen, or NH to hydroxyl hydrogen, are higher in energy by morethan 4 kcal/mol. The most important conclusion from this compu-tational survey is that the lowest energy zwitterionic structures aresignificantly higher in energy than the lowest canonical forms.Figure 2 also shows that the relative energies are similar whetherusing either DFT or ab initio methods.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
150 170 190 210 230 250 270 290
M
M-NH3
M-NH3-COM-NH3-CO2
a
b
Figure 1. (a) ESI and (b) CID mass spectra of PheSO3�. The CID for spec-
trum was carried out with a normalized collision energy of 25%
Figure 2. Low-energy conformations of the amino acid moiety ofPheSO3
� and their relative energies, in kcal/mol, computed at theB3LYP/6-31 + G* and MP2/6-311 + G** (in parentheses) level of theory
CID OF CANONICAL AMINO ACIDS AND LACTONES
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155
Dissociation of PheSO3�
The CID spectrum of PheSO3� is shown in Fig. 1(b). The major frag-
ments observed are readily assigned as resulting from dissociationof the canonical amino acid backbone. In particular, the main dis-sociation processes all involve loss of NH3 to form an ion withm/z 227, which subsequently fragments by loss of either CO, toform m/z 199, or CO2, to form m/z 183. That the m/z 183 and199 ions that come from the M–NH3 anion are confirmed by twoadditional results. First, although m/z 183 and 199 are major prod-ucts observed at the energy used for CID in Fig. 1(b), the M–NH3
ion is the only product observed at very low energies, consistentwith a sequential dissociation. Second, CID of the isolated M–NH3
anion, generated either by CID during the ESI injection stage orby CID in the course of an MS/MS/MS experiment, verifies that itdissociates by loss of CO and CO2 in a nearly 1:1 ratio as observedin Fig. 1(b). Therefore, the loss of NH3 is the only pathway that canbe uniquely assigned to PheSO3
�. Direct loss of CO2, which hasbeen found previously for pyrolysis of gas-phase amino acids(Eqn 1),[20] is not observed upon activation of PheSO3
�.Although the structure of the M–NH3 product is not known,
the most likely products to consider are those that are stableproducts formed by minimal rearrangement. Therefore, the mostlikely products are either a cis-cinnamic acid or trans-cinnamicacid (CASO3
�), an α-lactone (αLSO3�), or the phenyl acrylate
(PASO3�). Other products are possible but would require more
extensive rearrangement.Collision-induced dissociation of deprotonated phenyl alanine
is known to result in formation of cinnamic acid.[44–46] Similarly,dissociation of PheSO3
� by elimination across the ethylene back-bone would result in the formation of a cinnamic acid, CASO3
�,as shown in Eqn 4. However, multiple lines of evidence argueagainst the cinnamic acid. First, ammonia loss from d3-labeledPheSO3
�, formed by H/D exchange using a mixture of D2O/CD3CO2D as the solvent, occurs by loss of ND3 only, and loss ofND2H, NDH2, or NH3 is not observed to any detectable extentat any collision energy.
This rules out the possibility of elimination across the back-bone as shown in Eqn 4. It is also evidence against other morecomplicated rearrangement processes involving non-amino acidportions of the molecule.
However, cinnamic acid formation from PheSO3� does not re-
quire dissociation across the backbone. An alternate mechanism,as shown in Eqn 5, proceeds via a zwitterionic intermediate.
Therefore, we have examined the CID behavior of authentictrans-cinnamic acid and cis-cinnamic acid to compare with theproduct obtained from PheSO3
�. For this experiment, sulfonatedtrans-cinnamic acid was formed by ESI of an aqueous solution ofthe commercially available sulfonyl chloride, and the cis-cinnamicacid was formed in situ by CID of the corresponding ethyl ester. Acomparison of the CID spectra of the m/z 227 product obtainedfrom PheSO3
� and the authentic cinnamic acids, measured witha normalized collision energy of 34%, is shown in Fig. 3. Whereas
Figure 3. CID spectra of m/z 227 ions obtained (a) by CID of PheSO3�,
and from sulfonated (b) cis and (c) trans-cinnamic acid
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156
all ions lose CO2 as the major fragmentation pathway, significantdifferences are evident. For example, the CASO3
� ions do not loseCO to any appreciable extent, and lose only SO2 (to formm/z 163)in addition to losing CO2. Moreover, although the m/z 183 ionthat corresponds to PheSO3
�–NH3–CO2 is found to lose SO2 uponCID (vide infra), the CASO3
� ions undergo facile loss of CO2 andSO2 to form m/z 119 in much higher yield than is found for theM–NH3 ion. Finally, although the authentic CASO3
� ions dissoci-ate by loss of CO2, they do so only at energies much higher thanthose needed for dissociation of PheSO3
�. The amounts of them/z 163 and m/z 119 products observed in Fig. 3(a), 3(b), and 3(c) indicate that CASO3
� constitutes at most about 5% of theM–NH3 product.As shown in Eqn 6, the zwitterionic intermediate could also re-
arrange through nucleophilic attack at the ipso-position followedby a Grob-like fragmentation to give PASO3
�.
Although PASO3� is likely a relatively stable product, it can be
ruled out for multiple reasons. First, it is inconsistent with the CIDresults, in that it cannot readily lose either CO or CO2. Second,formation of the phenyl acrylate would require a high-energy,non-aromatic transition state (vide infra). Ultimately, the forma-tion of PASO3
� was ruled out by using an authentic sample.The primary dissociation pathway of PASO3
� is loss of theacryloyl group (CH2¼CHCO) to form the phenoxyl radical, lossof CO or CO2 does not occur at all (Supporting Information).In contrast, formation of the α-lactone is consistent with all
data. Formation of the lactone, either by a concerted loss ofNH2–H or through a zwitterionic intermediate (Eqn 7), involvesloss of only the exchangeable protons, and is therefore consis-tent with the results for the deuterated ions.
Loss of CO like that observed here is a well-known decompo-sition pathway for α-lactones,[24–36] and therefore, loss of COfrom the M–NH3 anion is consistent with the presence of theα-lactone. Although decarboxylation is generally not considereda decomposition pathway for that type of structure, it is not
unprecedented,[47] and computational studies described hereinpredict that it is expected to occur for αLSO3
�.Based on these results, we conclude that the M–NH3 ion found
upon CID of PheSO3� is most likely the α-lactone, αLSO3
�. Al-though α-lactones are nominally high-energy reactive species,they have previously been proposed as products in the CID ofα-substituted carboxylates in mass spectrometry experiments,[48–51]
which is essentially how it is shown being formed via the zwitter-ion in Eqn 7. The difference between the previous studies and thiswork is that the α-lactone formed upon fragmentation charge-remote fragmentation of PheSO3
� is ionic, and therefore detect-able by mass spectrometry. Previous studies of halogenatedcarboxylates[48–51] have inferred α-lactone structures based onthe observation of halide products.
Dissociation of the α-lactone, αLSO3�
As described in the aforementioned section, the PheSO3�–NH3
product, assigned to be the α-lactone (αLSO3�), dissociates by
loss of either CO or CO2. Loss of CO from α-lactones is well-knownand predicted to occur with a barrier of about 28–30 kcal/mol[52]
to form the corresponding aldehyde or ketone (Eqn 3). The CO-loss product obtained upon CID of αLSO3
� in this work is foundto undergo further dissociation by loss of 29 mass units, whichwe assign to HCO as shown in Eqn 8.
Dissociation to form the benzyl radical product, BzSO3�, is also
observed upon CID of control substrates, such as sulfonatedphenylpropionic acid or phenethylamine (Eqn 9), and so it isnot surprising that it is also observed for the aldehyde.
The loss of CO2 has not been reported previously for the gas-phase decomposition of α-lactones, although it has been foundto occur, along with decarbonylation, upon photolysis of thebis-n-butyl-substituted α-lactone in solution,[47] forming the ole-fin (Eqn 10).
By comparing CID spectra of the m/z 183 product with an au-thentic sample (Supporting Information), we have confirmedthat the product formed upon decarboxylation of αLSO3
� is sim-ilarly the corresponding styrene (Eqn 11).
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In summary, we have found that the dissociation of the gas-phase amino acid, PheSO3
�, results in the formation of an α-lactone, αLSO3
�, which dissociates by decarbonylation to formthe aldehyde, and by decarboxylation accompanied by hydrogenshift to form the styrene.
DISCUSSION
The results described in the previous section are the first studiesof the dissociation of a simple (non-N-substituted), canonicalamino acid in the gas phase. The dissociation pathway for theamino acid is similar to that observed previously for substitutedalanine[23] and other substituted carboxylic acids,[24–36] but isvery different from what has been reported for glycine deriva-tives.[20–22] The difference in dissociation pathways between gly-cine and alanine derivatives has been noted previously[22] buthas not been explained satisfactorily.
That the decomposition of PheSO3� involves only the amino
acid moiety and not the SO3� group is evidence that the sulfo-
nate charge does not participate in the dissociation processes.Fragmentation of the SO3
� group is not even observed for anyof the dissociation products, except for the sulfonated styreneshown in Eqn 11, which dissociates by loss of SO2. Therefore,the experimental results indicate that the sulfonate group isserving extensively as a charge carrier, and does not significantlyaffect the possible dissociation pathways. Although the dissocia-tion pathway, loss of NH3, is the same as what is found fordeprotonated phenylalanine,[44–46] the product formed with thation is deprotonated cinnamic acid. Control experiments in thiswork show that cinnamic acid is not formed upon dissociationof PheSO3
�. The mechanism proposed for deprotonated phenyl-alanine[44–46] involves intramolecular proton transfer to thecarboxylate and subsequent proton transfer to the amine, andtherefore, the charge center is extensively involved in the pro-cess. The lack of formation of cinnamic acid with PheSO3
�
suggests that the charge center is not participating in the disso-ciation process. Deprotonated tyrosine also undergoes CID byloss of ammonia,[53] possibly via the phenoxide ion.
Electronic structure calculations predict that the sulfonatecharge does not have a large effect on the energies of the reactionpathways for PheSO3
�. A comparison of the relative energies ofobserved and other possible products in the dissociation ofPheSO3
� and neutral phenylalanine, Phe, calculated at theMP2/6-311+G**//B3LYP/6-31+G* level of theory, is shown inTable 1. Table 1 shows that the relative energies for all the possibledissociation products for PheSO3
� and Phe are very similar, withdifferences for the primary process of less than 3 kcal/mol.
Amino acid dissociation
A potential energy surface for the dissociation of PheSO3�,
shown in Fig. 4, provides insight into the product selectivity. Be-cause CID is a kinetic process, the most important features of thepotential energy surface are the activation barriers. Therefore,
we have calculated the barriers for NH3 and CO2 loss fromPheSO3
� and Phe at the MP2/6-311 +G**//B3LYP/6-31 +G* levelof theory. Despite multiple attempts, we could not find any tran-sition states that directly connect the canonical structure of theamino acid with the expected deamination or decarboxylationproducts. However, insight into the mechanism was obtainedfrom multidimensional potential energy surface calculations(Supporting Information). We find that loss of NH3 or CO2 occurseffectively by dissociation of the zwitterionic structure, in thatproton transfer from the carboxylic acid to the amino groupoccurs very early in the process at energies well below thoserequired for dissociation.
Table 1. Computed reaction energies for dissociation path-ways of PheSO3
� and Phe1
Relativeenergies
Products PheSO3� Phe
Amino acid (reactant) 0.0 0.0ArCH2C(CO)CH+NH3 (α-lactone, Eqn 7) 47.0 44.6ArCH¼CHCO2H+NH3 (trans-cinnamicacid, Eqn 4)
17.7 16.4
ArCH2CH2NH2 +CO2 1.1 -4.6ArCH2CHO+CO+NH3 34.9 31.9ArCH¼CH2 +CO2 +NH3 15.9 11.4ArOC(O)CH¼CH2 +NH2 39.4 39.01Electronic energy differences between products and theamino acid reactants, computed at the MP2/6-311 +G**//B3LYP/6-31 +G* level of theory.
Figure 4. Calculated energetics for the dissociation of PheSO3�
(X¼SO3�) and Phe (X¼H). Values correspond to electronic energies and
geometries calculated at the MP2/6-311 + G**//B3LYP/6-31 + G* level oftheory (a) not a stable geometry; correspond to an amino acid geometrythe same as that for zwitterionic PheSO3
�; (b) computed utilizing a3 kcal/mol barrier for the reverse reaction, obtained from a relaxed sur-face scan; see Supporting Information
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Loss of NH3 from Phe to form the α-lactone is estimated tohave a barrier of about 3 kcal/mol higher than the reaction en-ergy, about 46 kcal/mol. The mechanism for ammonia elimina-tion involves an intramolecular SN2 reaction (Eqn 12), wherethe carboxylate displaces the amine leaving group in the zwitter-ionic structure. Therefore, the dissociation of PheSO3
� to formthe α-lactone is analogous to the elimination reactions of α-halocarboxylates that have been reported previously.[50,54–60] Inan ab initio study, Davidson and co-workers[61] found that thereis no barrier in excess of the dissociation energy for the forma-tion of the α-lactone from chloroacetate, whereas a small barrierwas calculated for the zwitterionic structure of Phe in this work.
The barrier for elimination of NH3 to form the cinnamic acid,not shown in Fig. 4, is calculated to be 68 kcal/mol, which ac-counts for why that process does not occur. However, given thatcharge-remote fragmentation is analogous to pyrolysis, it is notsurprising that cinnamic acid is not formed, considering that py-rolysis of alkylated amines does not occur by concerned elimina-tion of NH3, but results in the formation of imines.[62] Similarly,we have calculated the barrier for the formation of the phenylacrylate, PASO3
�, as shown in Eqn 6. Whereas for the neutralphenyl alanine, we find a concerted transition state for the rear-rangement accompanied by ammonia loss, the transition statefor the sulfonated version has the ammonia completely dissoci-ated, and is part of a stepwise process. In either case, the barrierfor phenyl acrylate formation is very high, computed to be about80 kcal/mol from the zwitterion, and 90–100 kcal/mol from thecanonical structure.Although loss of CO2 from the amino acid is not observed in
this work, it has been reported previously, and therefore, wehave carried out calculations to determine why it does not occurfor PheSO3
�. Decarboxylation of the zwitterionic structure of Pheis calculated to occur without a barrier in excess of the dissocia-tion energy, such that the net barrier for the reaction is deter-mined by the energies of the dissociation products. Therefore,loss of CO2 has a barrier of more than 60 kcal/mol. As shown inFig. 4, loss of CO2 would necessarily lead to formation of thezwitterionic product (the ammonium ylide) and not thephenethyl amine. Although formation of the amine is exother-mic, Alexandrova and Jorgensen[63] have noted that the barrierfor proton shift in the ylide is formally symmetry forbidden andis therefore expected to have a very high barrier. Consequently,the formation of the amine from glycine is essentially a sequen-tial, two-step process consisting of decarboxylation followed byproton transfer, with a proton transfer barrier of approximately45 kcal/mol for the ylide in aqueous solution.[63] The barrier forproton shift in the ammonium ylide formed upon decarboxyl-ation of Phe in the gas phase is calculated to be 25.6 kcal/mol,such that the overall barrier for the formation of the amine upondecarboxylation of Phe is calculated to be 86.4 kcal/mol. Thehigher barrier for proton shift computed for glycine is likelydue to solvent stabilization of the ammonium ylide. Interestingly,Alexandrova and Jorgensen also calculated a slight (~8 kcal/mol)barrier in excess of the dissociation energy for the decarboxylation
step. However, the presence of the excess barrier is also likely aconsequence of explicitly including solvation in the QM/MM ap-proach, and would not be expected in a gas-phase calculation.[64]
The barriers for decarboxylation obtained for glycine byAlexandrova and Jorgensen[63] and for Phe in this work aresignificantly higher than what has been reported previouslyfor the decarboxylation of canonical N,N-dimethylglycine orN-phenylglycine.[20,21,65] However, the ~42 kcal/mol barrierssuggested in the previous studies do not seem reasonable be-cause they are lower than the energies required for decarbox-ylation, which we calculate to be 52.3 and 59.6 kcal/mol fordimethylglycine and N-phenylglycine, respectively. Consideringthat decarboxylation leads initially to the ammonium ylide,and that rearrangement of the ylide to the amine occurs in asecond step and is formally symmetry forbidden, decarboxyl-ation should not occur with an activation energy less than thatrequired for the formation of the ammonium ylide.
In summary, although loss of CO2 from the amino acid can oc-cur to form the ammonium ylide, formation of the α-lactone ispredicted to be energetically preferred by about 20 kcal/mol.Consequently, that is the only process that is observed forPheSO3
�.
Dissociation of the α-lactone
In this study, we have also been able to investigate the dissocia-tion of the α-lactone, αLSO3
�. α-Lactones are highly reactive mol-ecules that have been proposed as short-lived intermediates in avariety of chemical reactions, often involving α-substituted car-boxylates[50,54–60] or α,β-unsaturated acids,[66,67] in the photolysisof cyclic peroxides[47,68–71] and α-halocarboxylic acids,[72] in theoxidation of ketenes,[73] or in the gas-phase dissociation ofα-substituted carboxylic[24–29] or in the decomposition of aminoacids.[23,74] They have also been proposed as intermediates in at-mospheric oxidation reactions[75,76] in mass spectrometry,[77–80]
and as products of the reaction of carbenes with CO2.[81–85] De-
spite being highly reactive, α-lactones have been investigatedspectroscopically by using matrix isolation,[82–84] gas phase,[86]
and solution-phase time-resolved IR.[85] There has long beena discussion regarding the electronic structure of α-lactones,and whether they are best considered to be cyclic, canonicalstructures[56,58–60] or zwitterionic,[55] although those discussionsgenerally relate to the structure in solution. The calculated struc-tures of gas-phase α-lactones have exceptionally long (~1.55 Å)and weak[87] Cα–O bonds, and even in the gas phase, there is con-siderable ionic character.[87]
α-Lactones are highly reactive, and react rapidly by polymeri-zation, by nucleophilic addition, or by decomposition.[88] Al-though an early photolysis study reported dissociation by lossof CO and CO2,
[47] more recent studies of α-substituted carboxylicacid pyrolysis that proceeds through α-lactone intermedi-ates[24,25,30–36] have reported dissociation only by loss of CO. Inthis work, we observe loss of both CO and CO2 from the α-lactone,in similar amounts (if anything, CO2 loss is slightly favored). As de-scribed earlier, loss of CO occurs to form the aldehyde, as ex-pected, whereas loss of CO2 results in the formation of thestyrene (Eqn 10).
Computed potential energy surfaces for the decompositionchannels are shown in Fig. 5. The barriers for CO loss from theα-lactone derived from PheSO3
� and Phe are predicted to beabout 30 kcal/mol, similar to that predicted for alkyl-substitutedα-lactones.[52] Determining the barrier for styrene formation is
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more difficult. The experimental observation that CO and CO2
are formed similarly indicates that the barriers for the two pro-cesses are similar. Therefore, a stepwise mechanism consistingof decarboxylation to form the carbene followed by hydrogenshift likely has a barrier that is too high to compete with CO loss(Fig. 5). Considering that decarboxylation of β-lactones results di-rectly in the formation of the olefin and occurs with moderateenergy barriers,[89,90] we explored the possibility of a mechanisminvolving the dyotropic shift[91–94] to the β-lactone[95] followedby decarboxylation (Eqn 13a). However, the search for the transi-tion state for α-lactone to β-lactone rearrangement did not give astructure for a dyotropic process, as shown in 10a, but insteadgave a structure that consists of 1,2-hydrogen shift without assis-tance of the carboxylate anion, as shown in Eqn 13b. The struc-ture shown in Eqn 13b was confirmed to be a saddle point byvibrational analysis, and an intrinsic reaction coordinate calcula-tion[96,97] finds that it is a transition state that connects theα-lactone with the styrene and CO2.
The 1,2-hydrogen shift mechanism shown in Eqn 13b is facili-tated by the high degree of polarization expected for the α-lactone(Eqn 14),[87] and by the fact that the β-cationic carboxylate (β(+)–CO2) that would result from hydride transfer is unstable with
respect to decarboxylation in the gas phase. The coupling of hy-drogen migration and leaving-group loss is analogous to whathas been proposed for the Schmidt reaction,[94] and has been ob-served previously in reactions such as the elimination of proton-ated ethers[98,99] and acetates.[100]
The computed barriers for decarboxylation of the α-lactoneare 32.1 kcal/mol for αLSO3
�, and 42.1 kcal/mol for the non-ionicsystem, indicating that the sulfonate is having a large effect onthe stability of the transition state. Because the charge of the sul-fonate group does not delocalize into the π-system of the ringvia conjugation, the stabilization of the transition state is likelyan inductive effect that stabilizes the formation of the benzyliccation. The effect would be expected to be stronger for an ionicgroup that is conjugated with the aromatic ring, such as an O�
group of a phenoxide (Eqn 15), which accounts for why thephenoxide ion obtained from deprotonating tyrosine loses NH3
and CO2 as the main dissociation channel, and an M–NH3–COion is not observed at all.[53]
The calculated barriers for decarboxylation and decarbonylationof the ionic and non-ionic substrates are consistent with the exper-imentally observed results. The large difference in barrier heightsfor the non-ionic lactone accounts for why loss of CO is generallythe only observed channel in pyrolysis experiments. However, forαLSO3
�, the computed barriers for loss of CO and CO2 are very sim-ilar, which accounts for why the products are observed in nearlyequal amounts. As shown in Eqn 15, the resonant interaction be-tween the charge site and the benzylic site in deprotonated tyro-sine is expected to strongly favor the CO2 loss channel. Finally,preliminary results with para-trimethylammonium-substitutedPhe find that the corresponding α-lactone dissociates only by lossof CO, consistent with electrostatic destabilization of the benzyliccation.
CONCLUSIONS
By using an ion with the charged moiety isolated from an aminoacid, we have been able to utilize mass spectrometry to investi-gate the chemical properties of a gas-phase amino acid. In thiswork, we have characterized the decomposition pathways for aphenylalanine derivative. The only observed pathway is loss ofammonia, to form an α-lactone. This reaction is similar to whathas been observed previously for pyrolysis of N-substituted ala-nines[23] and other α-substituted carboxylic acids,[24–36] but is in-consistent with what has been reported for pyrolysis of glycine
Figure 5. Calculated energetics for the dissociation of αLSO3� (X¼SO3
�)and the non-sulfonated derivative (X¼H). Values correspond to elec-tronic energies and geometries calculated at the MP2/6-311 + G**//B3LYP/6-31 + G* level of theory, unless indicated (a) not a stable geome-try; corresponds to the geometry of the lactone with the CO2 removed
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derivatives.[20–22] The main difference between the studies thatfind deamination[23] (including this work) and those that find de-carboxylation to occur[20–22] is our work and that of Al-Awadiet al.[23] involve a direct investigation of the gaseous amino acid,whereas Chuchani and co-workers have tried to generate theamino acid in situ by pyrolysis of the corresponding ethyl esters.Considering that the computational results in this work and byothers[63] have shown that decarboxylation of gaseous aminoacids to form the amine has a prohibitively high barrier, theproducts that correspond to decarboxylation of the amine arelikely formed via a pathway not involving the amino acid.The α-lactone that is formed from the amino acid in this work
undergoes dissociation by loss of CO and CO2. Although loss ofCO is expected, the loss of CO2 has generally not been reportedin reactions involving α-lactones. However, given the calculatedrelative barriers for CO and CO2 loss, decarboxylation should oc-cur, to some extent, in α-lactones containing a β-hydrogen thatcan shift during lactone ring-opening, similar to the reactionshown in Eqn 13b. It may be that the formation of the benzyliccation may facilitate the hydride shift reaction, although decar-boxylation has been observed for a completely aliphatic deriva-tive (Eqn 9).[47] The results of this work and that reportedpreviously for deprotonated tyrosine show that stabilization ofthe β-cation can affect the branching ratio for CO versus CO2
loss. Therefore, just as anionic stabilization of the transition statefavors CO2, the presence of a cationic group would be expectedto disfavor decarboxylation, and favor loss of CO, which is what isobserved in preliminary studies.Finally, the results of this study show that mass spectrometry
can be used to investigate the properties of canonical aminoacids. Whereas this is a new approach for the investigation ofgas-phase amino acids, the use of an inert, remote charge to in-vestigate neutral chemistry is not new and is similar, in principle,to the distonic ion approach used by Kenttämaa and co-workers[101] to examine the reactivity of aromatic radicals. Inthe same way, it should be possible to use mass spectrometryto investigate further the reactivity of gas-phase amino acids,and investigate the effects of structure and solvation.
Acknowledgements
This work was supported by the National Science Foundation(CHE11-11777). Calculations were carried out using the resourcesof the Center for Computational Studies of Open-Shell and Elec-tronically Excited Species (iopenshell.usc.edu), supported by theNational Science Foundation through the CRIF:CRF program. Wethank Profs. Anastassia Alexandrova and Nouria Al-Awadi for thehelpful comments, Dr. Will McGee for the help with the triple-quadrupole experiments, and Hari R. Khatri for the assistancewith the synthesis and characterization of the cis-cinnamic acid.
REFERENCES[1] Y.-J. Kuan, S. B. Charnley, H.-C. Huang, W.-L. Tseng, Z. Kisiel,
Astrophys. J. 2003, 593, 848.[2] L. E. Snyder, F. J. Lovas, J. M. Hollis, D. N. Friedel, P. R. Jewell, A.
Remijan, V. V. Ilyushin, E. A. Alekseev, S. F. Dyubko, Astrophys. J.2005, 619, 914.
[3] A. C. V. Johansson, E. Lindahl, J. Chem. Phys. 2009, 130, 185101.[4] C. J. Chapo, J. B. Paul, R. A. Provencal, K. Roth, R. J. Saykally, J. Am.
Chem. Soc. 1998, 120, 12956.[5] R. R. Julian, M. F. Jarrold, J. Phys. Chem. A. 2004, 108, 10861.[6] J. H. Jensen, M. S. Gordon, J. Am. Chem. Soc. 1995, 117, 8159.
[7] E. Kassab, J. Langlet, E. Evleth, Y. Akacem, J. Mol. Struct.(THEOCHEM). 2000, 531, 267.
[8] M. F. Jarrold, Annu. Rev. Phys. Chem. 2000, 51, 179.[9] T. S. Zwier, J. Phys. Chem. A. 2006, 110, 4133.[10] J. M. Bakker, L. M. Aleese, G. Meijer, G. v. Helden, Phys. Rev. Lett.
2003, 91, 203003.[11] J.-P. Schermann, Spectroscopy and Modelling of the Biomolecular
Building BlocksElsevier, Netherland, 2008.[12] Y. Hu, E. R. Bernstein, J. Chem. Phys. 2008, 128, 164311/1.[13] Y. Hu, E. R. Bernstein, J. Phys. Chem. A. 2009, 113, 8454.[14] R. Antoine, P. Dugourd, Phys. Chem. Chem. Phys. 2011, 13,
16494.[15] Y. K. Gao, F. Traeger, K. Kotsis, V. Staemmler, Phys. Chem. Chem.
Phys. 2011, 13, 10709.[16] H.-W. Jochims, M. Schwell, J.-L. Chotin, M. Clemino, F. Dulieu, H.
Baumgartel, S. Leach, Chem. Phys. 2004, 298, 279.[17] J. Adams, Mass Spectrom. Rev. 1990, 9, 141.[18] C. Cheng, M. L. Gross, Mass Spectrom. Rev. 2000, 19, 398.[19] J. Adams, M. L. Gross, J. Am. Chem. Soc. 1989, 111, 435.[20] A. Ensuncho, M. J. Lafont, A. Rotinov, R. M. Dominguez, A. Herize, J.
Quijano, G. Chuchani, Int. J. Chem. Kinet. 2001, 33, 465.[21] R. M. Dominguez, M. Tosta, G. Chuchani, J. Phys. Org. Chem. 2003,
16, 869.[22] M. Tosta, J. C. Oliveros, J. R. Mora, T. Cordova, G. Chuchani, J. Phys.
Chem. A. 2010, 114, 2483.[23] S. A. Al-Awadi, M. R. Abdallah, M. A. Hasan, N. A. Al-Awadi, Tetrahe-
dron. 2004, 60, 3045.[24] N. A. Al-Awadi, A. Kumar, G. Chuchani, A. Herize, Int. J. Chem. Kinet.
2001, 33, 612.[25] G. Chuchani, R. M. Dominguez, A. Rotinov, I. Martin, J. Phys. Org.
Chem. 1999, 12, 612.[26] L. R. Domingo, M. T. Picher, J. Andres, V. S. Safont, G. Chuchani,
Chem. Phys. Lett. 1997, 274, 422.[27] L. R. Domingo, M. T. Picher, V. S. Safont, J. Andres, G. Chuchani,
J. Phys. Chem. A. 1999, 103, 3935.[28] A. Rotinov, G. Chuchani, J. Andre, L. R. Domingo, V. S. Safont, Chem.
Phys. 1999, 246, 1.[29] V. S. Safont, V. Moliner, J. Andres, L. R. Domingo, J. Phys. Chem. A.
1997, 101, 1859.[30] G. Chuchani, R. M. Dominguez, Int. J. Chem. Kinet. 1995, 27, 85.[31] G. Chuchani, R. M. Dominguez, Int. J. Chem. Kinet. 1999, 31, 725.[32] G. Chuchani, R. M. Dominguez, A. Rotinov, Int. J. Chem. Kinet. 1991,
23, 779.[33] G. Chuchani, I. Martin, A. Rotinov, Int. J. Chem. Kinet. 1995,
27, 849.[34] G. Chuchani, I. Martin, A. Rotinov, R. M. Dominguez, J. Phys. Org.
Chem. 1993, 6, 54.[35] G. Chuchani, A. Rotinov, Int. J. Chem. Kinet. 1989, 21, 367.[36] G. Chuchani, A. Rotinov, R. M. Dominguez, J. Phys. Org. Chem. 1996,
9, 787.[37] Macromodel, version 9.6, Schrödinger, LLC, New York, NY, 2009.[38] J. Kong, C. A. White, A. I. Krylov, D. Sherrill, R. D. Adamson, T. R.
Furlani, M. S. Lee, A. M. Lee, S. R. Gwaltney, T. R. Adams, C.Ochsenfeld, A. T. B. Gilbert, G. S. Kedziora, V. A. Rassolov, D. R.Maurice, N. Nair, Y. Shao, N. A. Besley, P. E. Maslen, J. P. Dombroski,H. Daschel, W. Zhang, P. P. Korambath, J. Baker, E. F. C. Byrd, T. V.Voorhis, M. Oumi, S. Hirata, C.-P. Hsu, N. Ishikawa, J. Florian, A.Warshel, B. G. Johnson, P. M. W. Gill, M. Head-Gordon, J. A. Pople,J. Comput. Chem. 2000, 21, 1532.
[39] Gaussian 03, Revision B.03, M. J. Frisch, G. W. Trucks, H. B. Schlegel,G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. J. A. Montgomery, T.Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J.Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda,J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M.Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J.Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A.Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich,A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S.Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y.Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson,W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian, Inc.,Pittsburgh, PA, 2003.
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[40] J. E. Bartmess, “Negative Ion Energetics”. in: NIST Chemistry Webbook,NIST Standard Reference Database Number 69 (Eds: P. J. Lindstrom,W. G. Mallard), National Institutes of Standards and Technology:Gaithersburg, MD, 20899, http://webbook.nist.gov, (retrieved June2, 2015).
[41] T. T. Nha Tran, T. Wang, S. Hack, J. H. Bowie, Rapid Commun. MassSpectrom. 2013, 27, 1135.
[42] Z. Tian, A. Pawlow, J. C. Poutsma, S. R. Kass, J. Am. Chem. Soc. 2007,129, 5403.
[43] Z. Tian, X.-B. Wang, L.-S. Wang, S. R. Kass, J. Am. Chem. Soc. 2009,131, 1174.
[44] M. Eckersley, J. H. Bowie, R. N. Hayes, Int. J. Mass Spectrom. Ion Pro-cesses. 1989, 93, 199.
[45] S.-S. Choi, O.-B. Kim, Int. J. Mass spectrom. 2013, 338, 17.[46] A. M. Couldwell, M. C. Thomas, T. W. Mitchell, A. J. Hulbert, S. J.
Blanksby, Rapid Commun. Mass Spectrom. 2005, 19, 2295.[47] W. Adam, R. Rucktaeschel, J. Org. Chem. 1978, 43, 3886.[48] M. Baker, W. Gabryelski, Int. J. Mass spectrom. 2007, 262, 128.[49] T. S. Choi, J. Y. Ko, S. W. Heo, Y. H. Ko, K. Kim, H. I. Kim, J. Am. Soc.
Mass Spectrom. 2012, 23, 1786.[50] S. T. Graul, R. R. Squires, Int. J. Mass Spectrom. Ion Processes. 1990,
100, 785.[51] N. J. Rijs, R. A. J. O’Hair, Dalton Trans. 2012, 41, 3395.[52] L. R. Domingo, J. Andres, V. Moliner, V. S. Safont, J. Am. Chem. Soc.
1997, 119, 6415.[53] Z. Tian, S. R. Kass, J. Am. Chem. Soc. 2008, 130, 10842.[54] C. M. Bean, J. Kenyon, H. Phillips, J. Chem. Soc. 1936, 303.[55] W. A. Cowdrey, E. D. Hughes, C. K. Ingold, S. Masterman, A. D. Scott,
J. Chem. Soc. 1937, 1252.[56] E. Grunwald, S. Winstein, J. Am. Chem. Soc. 1948, 70, 841.[57] B. Strijtveen, R. M. Kellogg, Recl. Trav. Chim. Pays-Bas. 1987, 106,
539.[58] S. Winstein, J. Am. Chem. Soc. 1939, 61, 1635.[59] S. Winstein, R. B. Henderson, J. Am. Chem. Soc. 1943, 65, 2196.[60] S. Winstein, H. J. Lucas, J. Am. Chem. Soc. 1939, 61, 1576.[61] D. Antolovic, V. J. Shiner, E. R. Davidson, J. Am. Chem. Soc. 1988,
110, 1375.[62] Y. Hamada, H. Takeo, Appl. Spectrosc. Rev. 1992, 27, 289.[63] A. N. Alexandrova, W. L. Jorgensen, J. Phys. Chem. B. 2011, 115,
13624.[64] A. N. Alexandrova, personal communication ed., 2013.[65] J. R. Perez, M. Lorono, R. M. Dominguez, T. Cordova, G. Chuchani,
J. Phys. Org. Chem. 2008, 21, 402.[66] N. Pirinccioglu, J. J. Robinson, M. F. Mahon, J. G. Buchanan, I. H. Wil-
liams, Org. Biomol. Chem. 2007, 5, 4001.[67] S. Niwayama, H. Noguchi, M. Ohno, S. Kobayashi, Tetrahedron Lett.
1993, 34, 665.[68] W. Adam, J.-C. Liu, O. Rodriguez, J. Org. Chem. 1973, 38, 2269.[69] W. Adam, R. Rucktaeschel, J. Amer. Chem. Soc. 1971, 93, 557.[70] O. L. Chapman, P. W. Wojtkowski, W. Adam, O. Rodriguez, R.
Rucktaeschel, J. Amer. Chem. Soc. 1972, 94, 1365.[71] W. Adam, L. Blancafort, J. Org. Chem. 1996, 61, 8432.[72] S. Nishino, M. Nakata, J. Mol. Struct. 2008, 875, 520.
[73] M. Schmittel, H. von Seggern, Liebigs Ann. Chem. 1995, 1815.[74] J. Kenyon, H. Phillips, Trans. Faraday Soc. 1930, 26, 451.[75] S. A. Carr, D. R. Glowacki, C.-H. Liang, M. T. Baeza-Romero, M. A.
Blitz, M. J. Pilling, P. W. Seakins, J. Phys. Chem. A. 2011, 115, 1069.[76] H. G. Kjaergaard, H. C. Knap, K. B. Oernsoe, S. Joergensen, J. D.
Crounse, F. Paulot, P. O. Wennberg, J. Phys. Chem. A. 2012, 116,5763.
[77] D. Schroder, N. Goldberg, W. Zummack, H. Schwarz, J. C. Poutsma,r. R. Squires, Int. J. Mass Spectrom. Ion Processes. 1997, 165/166, 71.
[78] A. K. Y. Lam, R. A. J. O’Hair, Rapid Commun. Mass Spectrom. 2010,24, 1779.
[79] J. A. Wyer, L. Feketeova, N. S. Brondsted, R. A. J. O’Hair, Phys. Chem.Chem. Phys. 2009, 11, 8752.
[80] P. B. Armentrout, S. J. Ye, A. Gabriel, R. M. Moision, J. Phys. Chem. B.2010, 114, 3938.
[81] G. B. Kistiakowsky, K. Sauer, J. Am. Chem. Soc. 1958, 80, 1066.[82] D. E. Milligan, M. E. Jacox, J. Chem. Phys. 1962, 36, 2911.[83] S. Wierlacher, W. Sander, M. T. H. Liu, J. Org. Chem. 1992, 57, 1051.[84] W. W. Sander, J. Org. Chem. 1989, 54, 4265.[85] B. M. Showalter, J. P. Toscano, J. Phys. Org. Chem. 2004, 17, 743.[86] S.-Y. Chen, Y.-P. Lee, J. Chem. Phys. 2010, 132, 114303/1.[87] G. D. Ruggiero, I. H. Williams, J. Chem. Soc., Perkin Trans. 2. 2001,
733.[88] G. L’Abbé, Angew. Chem., Int. Ed. Eng. 1980, 19, 276.[89] I. Morao, B. Lecea, A. Arrieta, F. P. Cossio, J. Am. Chem. Soc. 1997,
119, 816.[90] R. Ocampo, W. R. Dolbier Jr., M. D. Bartberger, R. Paredes, J. Org.
Chem. 1997, 62, 109.[91] M. T. Reetz, Angew. Chem. Int. Ed. Engl. 1972, 11, 130.[92] M. T. Reetz, Angew. Chem. Int. Ed. Engl. 1972, 11, 129.[93] I. Fernandez, F. P. Cossio, M. A. Sierra, Chem. Rev. (Washington, DC,
U. S.). 2009, 109, 6687.[94] O. Gutierrez, D. J. Tantillo, J. Org. Chem. 2012, 77, 8845.[95] J. G. Buchanan, G. D. Ruggiero, I. H. Williams, Org. Biomol. Chem.
2008, 6, 66.[96] C. Gonzalez, H. B. Schlegel, J. Chem. Phys. 1989, 90, 2154.[97] C. Gonzalez, H. B. Schlegel, J. Phys. Chem. 1990, 94, 5523.[98] A. J. Ben, M. Karni, Y. Apeloig, A. Mandelbaum, Int. J. Mass
Spectrom. 2003, 228, 297.[99] N. Morlender-Vais, A. Mandelbaum, J. Mass Spectrom. 1997, 32,
1124.[100] I. Kuzmenkov, A. Etinger, A. Mandelbaum, J. Mass Spectrom. 1999,
34, 797.[101] R. L. Smith, K. K. Thoen, J. J. Nousiainen, E. D. Nelson, H. I.
Kenttamaa, J. Am. Chem. Soc. 1996, 118, 8669.
SUPPORTING INFORMATION
Additional supporting information may be found in the onlineversion of this article at the publisher’s web-site.
D. KOIRALA, S. R. KODITHUWAKKUGE AND P. G. WENTHOLD
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Reactivity of 3- and 4-pyridinylnitrene-n-oxide radical anions
Damodar Koirala a, James S. Poole b, Paul G. Wenthold a,*aDepartment of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, United StatesbDepartment of Chemistry, Ball State University, Muncie, IN, United States
A R T I C L E I N F O
Article history:Received 1 May 2014Received in revised form 4 July 2014Accepted 4 July 2014Available online 9 July 2014
Keywords:Flowing afterglowIon–molecule reactionsKineticsPyridine-n-oxidesNitrene radical anions
A B S T R A C T
Ion–molecule reactions in a flowing afterglow are used to examine the electronic structure of 3- and4-pyridinylnitrene-n-oxide radical anions. Reactions with nitric oxide are generally similar to thosereported previously for other aromatic nitrene radical anions. In particular, phenoxide formation bynitrogen–oxygen exchange is observed with both isomers. Oxygen atom abstraction by NO is alsoobservedwith both isomers. Very significant differences in the reactivity are observed in the reactions ofthe two isomers with carbon disulfide. The reactivity of the 3-n-oxide isomer with CS2 is similar to thatobserved previously for nitrene radical anions, and reactions of the n-oxide moiety are not observed,similar towhat is expected based on solution chemistry. The 4-n-oxide isomer, however, undergoesmanyreactions, including oxygen atom and oxygen ion transfer and sulfur–oxygen exchange, that involve then-oxide oxygen. The increased reactivity of the oxygen is attributed to increased charge density at theoxygen due to pi electron donation of the nitrene anion in the para position.
ã 2014 Elsevier B.V. All rights reserved.
1. Introduction
Nitrene radical anions are a fascinating class of hypovalent ions.Despite their unusual electronic structures, isoelectronic withcarbene radical anions [1–6], they are surprisingly easily generatedin mass spectrometry, cleanly and in high abundance, by simpleelectron ionization of azide-substituted precursors [7–20].Although azides are considered potentially explosive, given therecent interests in azides as reagents in “click” chemistry [21–24]they are commonly being synthesized and utilized in chemicalapplications, and, inprinciple, anyof these derivatives could be usedas precursors of nitrene radical anions. However, despite how easilythe ions are to form, few studies of nitrene radical anions have beenreported, and many of those reported have focused on the use ofthe anions as precursors for photoelectron/photodetachmentspectroscopy studies of the corresponding nitrenes [9–14].
Nevertheless, some studies have addressed issues of electronicstructures of nitrene radical anions. The simplest nitrene radicalanion, HN�, has been well-studied experimentally [25–31] andcomputationally [32]. Isoelectronic with OH, the HN� ion is foundto have a 2P electronic ground state. Ellison and co-workers [14]discussed the electronic structure of methyl nitrene radical anion,CH3N�, which has a 2E ground state, but, like CH3O, undergoes aJahn–Teller distortion [33,34].
[TD$INLINE]The electronic structures of phenylnitrene anions have been
discussed in the context of photoelectron [12,13] and photo-detachment [9–11] spectroscopy studies, and have been examinedby electronic structure calculations [13]. The planar phenylnitreneanion, PhN�, has an electronic structure that consist of threeelectrons in the two non-bonding molecular orbitals (NBMOs) ofPhN, which consist of an in-plane, s orbital, and a benzylic-likep orbital (Fig. 1). Unlike the case in C3v methylnitrene, the orbitalsin PhN are not degenerate, and therefore there are two possibleelectronic states that can be created, corresponding to s2p (2B2)andp2s (2B1). For substituted systems where the symmetry of thesystem is reduced to Cs, the electronic states correspond to 2A00 and2A0 , respectively. B3LYP calculations with large basis setspredict that the ground states of PhN and chloro-substitutedderivatives [13] are all p2s, with the s2p states lying approxi-mately 0.5 eV higher in energy.
Benzoylnitrene radical anion, BzN�, has been examinedcomputationally and experimentally [18–20]. Like PhN�, theground state of BzN� is predicted to be p2s (2A0), but the relativeenergy of the s2p state (28 kcal/mol) is predicted [18] to be muchhigher than that in PhN�, reflecting a greater preference for the
* Corresponding author. Tel.: +1 765 494 0475.E-mail address: [email protected] (J.S. Poole).
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International Journal of Mass Spectrometry 378 (2015) 69–75
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163
carboxylate-like anion over having the charge localized on anitrogen s orbital. Chemical reactivity of the BzN� is consistentwith the open-shell structure. In particular, reaction with NOresults via radical–radical coupling to give nitrogen/oxygenexchange (Eq. (1)), leading to the formation of benzoate anionand N2.
(1)
[TD$INLINE]In this study, we report an investigation of the reactivity of the
3- and 4-pyridinylnitrene-n-oxide radical anions, 3PNO� and4PNO�, respectively. The pyridine-N-oxide is an interestingstructural motif. The formal pyridinium can nominally be viewedas an
[TD$INLINE]electron-acceptor, but the adjacent oxide can act as ap donor, suchthat the electronic effect of the N-oxide is dependent on the extentof benefit that can be created, via electron donation or acceptance.However, the resonance effect of the n-oxide can only occur withthe nitrene nitrogen at the 4-position, and not at the 3-position.Moreover, for 4PNO�, there are two types of stabilization that canbe envisioned to result from interaction between the nitreneradical anion and the N-oxide, shown in Fig. 2. In thep2s state, thepyridinium can serve as an electron
pair acceptor, and the system can potentially be stabilized bycontribution from the quinone-like structure. Similarly, the s2pstate can be stabilized by radical delocalization, creating a highlystable nitroxyl radical. Electron delocalization in either state issuch that it eliminates the formal charge separation of then-oxides, which could provide additional stabilization.
We have carried out a computational study of the electronicstructures of3PNO� and 4PNO�, and an experimental investigationof their reactivity. Surprisingly, despite the possible interactionsbetween the nitrene radical anion andN-oxide in 4PNO�, wedonotfind significant differences in the computedelectronic structures orreactivities of the two isomers in the reactionwith NO. However, adramatic difference between the ions is found in the reaction withCS2, as the4-isomerundergoesreactivity that isnotobservedfor the3-isomer, attributed to differences in accessibility of the oxide endof the ion toward reaction. The results indicate that the resonance
interaction between the nitrene anion center and the n-oxideincreases the nucleophilicity of the oxygen in the n-oxide moiety.
2. Methods
2.1. Experimental procedures
The flowing afterglow used in the investigation has beenpreviously described [35]. Briefly, 3PNO� and 4PNO� radicalanions are generated in upstream end of flow tube by electronionization of corresponding azide precursor. The ions are cooled toambient temperature (298K) and carried downstream by heliumbuffer gas (0.400 Torr, flow (He) = 190 STP cm3/s), and are allowedto undergo ion–molecule reactionwith neutral reagent vapors. Theproduct ions are sampled through a 1mm orifice into a low-pressure triple–quadrupole mass filter and detected with anelectron multiplier. Reactions with mass selected ions can becarried out in the second quadrupole (Q2). Reactions in Q2 wereused to verify the reaction products observed in the flow tube. Inthese experiments, the Q2 dc pole offset was kept very low (�1–2V, laboratory frame) tominimize the possibility of translationallydriven reactions. The energy dependencies of the observedreaction products were examined to ensure that their intensitiesare maximized at nearly thermal collision energies and drop off athigher energies as expected for exothermic processes.
Reaction kinetics are determined by monitoring the depletionof reactant ion as a function of neutral flow rate for sampleintroduced at a fixed distance from the nose cone. Pseudo-firstorder reaction rate constants, krxn, are obtained from a logarithmicplot of ion depletion vs reagent flow rate. Reaction rates arereported as reaction efficiencies (eff), which are the ratios of themeasured rate constant to the collisional rate constant,kcoll, calculated by using the parameterized-trajectory methoddescribed by Su and Chesnavich [36]. Absolute uncertaintiesin measured rate constants are estimated to be �50%. Branchingratios in reactions with multiple observed products aredetermined by measuring the branching ratios at multipleneutral flow rates, and extrapolating to zero reagent flow.Uncertainties in branching ratios are estimated to be �10% onan absolute basis.
[(Fig._2)TD$FIG]
Fig. 2. Resonance structures of the P2s and s2P states of 4PNO�.
[(Fig._1)TD$FIG]
Fig. 1. Non-bonding molecular orbitals in phenylnitrene.
70 D. Koirala et al. / International Journal of Mass Spectrometry 378 (2015) 69–75
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2.2. Materials
The azidopyridine-n-oxide precursors were prepared usingpublished procedures [37–39]. The synthesis and characterizationof the samples used in this work has been reported previously [40].Other materials were obtained from commercial suppliers andused as received.
2.3. Computational methods
Structures and energies of the p2s and s2p states of 3PNO�
and 4PNO�, NO, CS2 and possible reaction products werecalculated at the B3LYP/6-31 +G* level of theory. Reaction energiescorrespond to differences in electronic energies between reactantsand products, and are not corrected for zero-point energies orthermal energy differences. Calculations were carried out by usingQChem [41], using the resources of the Center forComputational Studies of Open-Shell and Electronically ExcitedSpecies (iopenshell.usc.edu).
3. Results and discussion
In this section, we report the results of flowing afterglowstudies of the reactivity of 3PNO� and 4PNO� with nitric oxide(NO) and carbon disulfide (CS2). These reagents have been shownpreviously [18] to form characteristic products with nitrene radicalanions. For example, the N–O transfer that occurs in the reaction ofBzN� with NO (Eq. (1)) also occurs with PhN�, resulting in theformation of phenoxide anion [18]. With CS2, aromatic nitreneanions have been found to react by addition, and by C and CSabstraction to form S2� and S�, respectively. Similar reactionsshould be expected for 3PNO� and 4PNO�.
3.1. Ion formation
The 3PNO� and 4PNO� radical anions are formed in high yieldsby dissociative electron attachment to the corresponding azides,with m/z 108 (Eq. (2)).
(2a)
[TD$INLINE]
(2b)
[TD$INLINE]The most significant impurity observed in these experiments
(with both isomers) is an ionwithm/z 92, which is presumably thenon-oxidized pyridinylnitrene radical anion. The relative signal ofthem/z 92 ion is variable from day to day, but decreases with timeduring the course of an experimental run, indicating that it mostlikely results from ionization of a non-oxidized azidopyridineimpurity.
3.2. Ion electronic structures
Before addressing the reactivity results, we first consider thecomputed electronic structures of the ions. As described in the
introduction, aromatic nitrene radical anions can have either p2sor s2p electronic structures. Previous studies of substitutedphenylnitrene anions [13] have found that the p2s states arefavored. However, as shown in Fig. 2, resonance interactionswithinthe states could affect the relative energies.
At the B3LYP/6-31 +G* level of theory, both 3PNO � and 4PNO�
are predicted to have robust p2s ground states. The 2B1 state of4PNO� is calculated to be 13.2 kcal/mol lower in energy than the2B2 state, whereas the 2A0 state of 3PNO� is predicted to be16.0 kcal/mol lower in energy than the 2A00 state. The smalldifference in state energies of the sp2 can likely be attributed tothe preferential stability of the nitroxyl radical (Fig. 2), but thatstabilization is not large enough to overcome the benefits ofcharge delocalization. Nonetheless, the presence of the n-oxidegroup is not calculated to alter the ground state of the nitreneanion.
3.3. Reactivity studies
The results of our reactivity studies of ions 3PNO� and 4PNO�
are shown in Table 1. Overall, there are no measurable differencesin the rates of the reactions for the two isomers. The efficiencies ofthe reactions with NO (approximately 0.2) are much higher thanthose found for reaction with CS2, but similar to that reportedpreviously for the reaction of NO with BzN� (eff = 0.15) [18].Although there are no differences in the reaction rates for the twoisomers with these reagents, there are very large differences in theobserved products. In the sections below, we consider thedifferences in the products that are formed.
3.3.1. Reaction with NOWith nitric oxide, both 3PNO� and 4PNO� are observed to
undergoN–O exchange, similar towhat is observedwith PhN� andBzN� [18]. Many additional products are observed for the reactionof 3PNO� with NO, but, as shown in Table 1, they are assigned tosecondary fragmentation of the phenoxide anion. Possible productstructures are shown in Eq. (3)
(3).
[TD$INLINE]A notable difference in the reactivity of 3PNO� and 4PNO�with
NO is that reaction with 4PNO� leads to significant amount ofadduct ion, whereas only a trace is observed with 3PNO�. NOaddition was not reported for either PhN� or BzN� [18], and isgenerally associated with reactions of closed-shell anions [42,43],Unfortunately, the structures of the NO adducts are not known.However, charge distribution calculations reported below find thatthere is more charge on the oxygen in 4PNO� than in 3PNO�,which raises the possibility that the difference in the extent ofadduct formation is due to 4PNO� forming an adduct at theoxygen, as opposed to at the nitrogen.
D. Koirala et al. / International Journal of Mass Spectrometry 378 (2015) 69–75 71
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Both n-oxide anions are found to react with NO by oxygen atomtransfer to form the pyridinylnitrene radical anion, as shown for4PNO� in Eq. (4). Although the branching ratios for these reactionsin the flow tube cannot be determined due to the presence ofimpurity in ions, they were verified as products by using thereaction of NO with mass-selected ions in Q2.
(4)
[TD$INLINE]If the N�O BDEs in 3PNO� and 4PNO� are similar to that in
pyridine-n-oxide (approximately 72 kcal/mol) [44], then theoxygen atom transfer reactionwould be essentially thermoneutral.Alternatively, given that the reaction is carried out in Q2, albeitunder very low energy conditions, the oxygen atom transfer couldbe slightly endothermic and translationally driven.
3.3.2. Reaction with CS2Although the rates at which 3PNO� and 4PNO� react with CS2
are similar, there are significant differences between the reactionproducts. In particular, some products observed with 4PNO�
involve loss of the oxygen atom, and these products are notobserved with 3PNO�. One product involving the oxygen isobserved at m/z 124, which is 16Da higher than the reactant. Themost likely assignment for this product is that it arises fromaddition of sulfur and loss of oxygen, i.e., a sulfur–oxygenexchange. The resulting products would be the n-sulfide andcarbonyl sulfide, OCS.
The ion signal atm/z92 is also likelydue to the reaction involvingoxygen. As in the reaction with NO, we are unable to quantify theyield of the m/z 92 product due to background presence of thepyridinylnitrene radical anion. However, considering 4PyN� is alsoobservedtoreactwithCS2, it appears thatm/z92 is, in fact, formedinrelatively high yield (comparable to the yield of S2�). This is alsoindicated by the results on “clean” days, where the mass spectrumincludesminimal amounts of the impurity. The formation ofm/z 92in the reaction of 4PNO� with CS2 was also confirmed by usingmass-selected ions in Q2.
The identity of them/z 92 is not known unequivocally. Althoughthe ion with m/z 92 could be the pyridinylnitrene radical anion,there is a second possibility. The ion CS2O� is isobaric with thepyridinylnitrene radical anion, and could, in principle be formed byoxygen-anion transfer with 4PNO�, as shown in Eq. (5). Ourexperimental results suggest both structures are present.
(5)
[TD$INLINE]Evidence for the formation of CS2O� comes from the isotopic
distribution of the product. Fig. 3 shows amass spectrum of 4PNO�
taken on an exceptionally clean day (with minimal m/z 92contamination), with and without the addition of CS2. Thebackground signal of m/z 92 in the spectrum is less than 10kcps.However, upon addition of CS2, the signal increases to nearly100kcps. Most importantly, the signal at m/z 94 also increases, tonearly 8% of them/z 92 signal, which agrees well with the value of9% expected for CS2O�. Therefore, the M +2 isotope peak indicatesthat there is sulfur present in the m/z 92 ion.
However, reactivity evidence suggests the presence of 4PyN� aswell. Fig. 4 shows the mass spectra for reaction of m/z 92 ion,formed by reaction of 4PNO� with CS2, with NO. The formation ofm/z 94 is clear evidence for the presence of a nitrene radical anion,4PyN�.
Computationally,bothreactionsarecalculatedtobeenergeticallyfavorable. At the B3LYP/6-31+G* level of theory, the formation ofCS2O� and the triplet pyridinylnitrene (Eq. (5)) is computed to beexothermic by 72kcal/mol, or more than 3eV! For oxygen atomtransfer, there are multiple thermochemically accessible pathways.Direct transfer to formCS2O (Eq. (6a)) is computed to be exothermicby 7.7 kcal/mol. A second pathway, shown in Eq. (6b), involves theformation of CO+ triplet S2, and is computed to be exothermic by9.9 kcal/mol.
(6a)
[(Fig._3)TD$FIG]
Fig. 3. The m/z 92 region of the mass spectra of 4PNO� before (dashed) and after(solid) addition of CS2.
Table 1Reaction efficiencies and product branching ratios for reactions of 3PNO� and4PNO� with NO and CS2
Reagent Result Ion assignment 3PNO– 4PNO–
NO Effa 0.21 0.20m/z 138 [M+NO]� <1% 18%m/z 110 [M+NO�N2]� 44% 82%m/z 82 [M+NO�N2�CO]� 21% Nb
m/z 80 [M+NO�N2�NO]� 20% Nb
m/z 61 [M+NO�C3H3�N2]� 14% Nb
m/z 92 [M+NO�NO2]� Yc Yc
CS2 Effa 0.03 0.02m/z 152 [M+CS2]� 83% 77%m/z 64 S2� 15% 15%m/z 32 S� <1% <1%m/z 58 SCN� 2% 2%m/z 124 [M+CS2–CSO]� Nb 7%m/z 92 [M�O]� Nb Yc
a Reaction efficiency, which corresponds to kexp/kADO.b Not observed for this isomer.c Product is observed in Q2, but the flow tube branching ratio cannot be
determined due to the presence of impurities.
72 D. Koirala et al. / International Journal of Mass Spectrometry 378 (2015) 69–75
166
[TD$INLINE]
(6b)
[TD$INLINE]Regardless of which reaction occurs, oxygen atom transfer to
form4PyN�or oxygen ion transfer to formCS2O�, theyboth involvereaction at the oxygen. Deoxygenation of tertiary-amine-n-oxidesin solution [45–47] has been proposed to occur by a mechanismsuch as that shown in Scheme 1, leading to the formation of thedithiiranone as in Eq. (6a). In solution, the dithiiranone can behydrolyzed to CO2 +HSSH [45] or can be utilized as a sulfur transferreagent [48].
In the gas phase, direct reaction of oxygen atom with CS2 givesCS+ SO as the major products [49]. However, CO has also beenobserved in the reaction [50], indicating that CO+S2 is a possibledecomposition pathway of CS2O. In fact, CO+S2 is energetically themost favored pathway [49] but is slow because there is a slight(6.6 kcal/mol) barrier [49] for the initial formationofCS2O.However,thisbarriercanbeovercomeinthereactionofCS2with4PNO�bytheenergy releasedupon formationof the initial ion/molecule complexin combination with the oxygen atom transfer exothermicity.Therefore, oxygen atom transfer to form CO and S2 products shouldbe energetically accessible for 4PNO�. Formation of CS + SO isapproximately3 eV less favorable [49] than formationofCO+S2andis therefore highly endothermic in the reaction of 4PNO� with CS2.
As noted above, sulfur–oxygen exchange is also observed in thereaction of CS2 with 4PNO�. The mechanism of sulfur–oxygenexchange likely involves a first step similar to that for oxygen atomor ion transfer, addition of the oxygen to the center carbon of CS2,as shown in Scheme 2. However, instead of forming the disulfidebond as in dithiiranone formation, a N��S bond is formed.Alternatively, homolytic cleavage of the N��O bond in the CS2adduct would result in formation of CS2O�. Therefore, all threereaction pathways (oxygen atom transfer, oxygen ion transfer,oxygen–sulfur exchange) occur addition of the oxygen to CS2.
A small amount of NCS� product is observed in the reaction ofCS2 with both 3PNO� and 4PNO�. NCS� has been observedpreviously in reactions of CS2 with closed-shell, nitrogen-basedanions [43,51,52], but has not been reported previously forreactions of open-shell anions. The mechanism is presumablysimilar to that shown in Scheme 2, although occurring at thenitrogen. The other products observed with CS2 (S�, S2� and CS2adduct) are similar to those observed previously with nitreneradical anions [18].
3.4. Comparison of isomers: oxygen nucleophilicity
There are significant differences in the reactions that occurbetween ions 3PNO� and 4PNO� and CS2. Specifically, 4PNO�
undergoes sulfur–oxygen exchange and oxygen-atom and/oroxygen-anion transfer reactions that are not observedwith 3PNO�.The common feature of these reactions is that they all involveinitial nucleophilic attack of the oxygen in the anion at the carbonof the carbon disulfide. The fact that 4PNO� undergoes reaction atthe oxygenwhereas 3PNO� does not reflects important differencesin their electronic structures.
The best example that illustrates the electronic structuredifferences between the ions is the oxygen-transfer reactionwith CS2. Although carbon disulfide is known to deoxygenatetertiary-amine-n-oxides [45], the reaction is generally notobserved with aromatic-n-oxides. Therefore, the lack of oxygentransfer with 3PNO� is consistent with the expectation that thesubstituent in themeta position does not interact with the n-oxidemoiety. Similarly, the nitrene anion para to the n-oxide can serve asa p-donor, as shown in Fig. 2, which makes the oxygen in the p2sstatemore nucleophilic. Hammett analysis of the deoxygenation ofaniline-n-oxides in solution has shown [45] that the reaction isfavored by strong electron donating substituents, which increasethe nucleophilicity of the oxygen. Apparently, the nitrene radicalanion is such a strongp-electron donor that it even enables oxygentransfer in the normally inert aromatic-n-oxides.
The difference in the reactivity cannot be attributed todifferences in overall thermochemistry for the reactions. All of
[(Fig._4)TD$FIG]
Fig. 4. The m/z 92 region of the mass spectra of 4PNO� reaction with CS2 (dashed)and after addition of NO (solid).
[(Scheme_1)TD$FIG]
Scheme 1.
[(Scheme_2)TD$FIG]
Scheme 2.
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the reactions observed for 4PNO� are also computed to beexothermic for 3PNO�, although they do not occur. For example,the sulfur–oxygen exchange reaction observed for 4PNO�
(Scheme 2) is computed to be exothermic by 45kcal/mol. Similarly,the sulfur–oxygen exchange reaction for 3PNO� is computed to beexothermic by 37kcal/mol, but does not occur at all. Therefore, thedifference in reactivity is more likely due to kinetic differences thatresult from differences in electronic structure.
The increased nucleophilicity of the oxygen in 4PNO� apparentin the reactivity studies is consistent with the computed chargedistributions in the p2s states. The B3LYP/6-31 +G* computedMullikin charges at the heavy atoms in 3PNO� and 4PNO� areshown in Fig. 5.
Although both ions have increased charge density at the oxygenthan is found in pyridine-n-oxide there are some differences incharge densities at the carbon atoms, the most significantdifference is for the oxygen, where the calculated charge is largerin in 4PNO� than in 3PNO�, which accounts for the increasedreactivity at that site. Increased reactivity at the oxygen accountsfor the observation of either oxygen atom (Eq. (6)) or oxygen ion(Eq. (5)) transfer with CS2.
As suggested above, the difference in the charge density at theoxygen may also account for the difference in the extent of adductformation with nitric oxide. This is most likely the case if theadduct is an electrostatic complex. However, as noted, thestructure of the adduct is not known. Aside from the extent ofadduct formation, there is little difference in the reactivity of withnitric oxide. The differences in the observed products can beattributed to differences in the ability of the resulting phenoxideion to fragment.
4. Conclusion
The reactivity of 3PNO� and 4PNO�, particularlywith CS2, showthat there are significant differences in their electronic structures,which can be understood as resulting from the resonanceinteraction between the nitrene anion and the n-oxide moietyin 4PNO�, and the lack thereof in 3PNO�. In the p2s state, themonovalent nitrogen anion is a strong p electron donor, whichincreases the charge density on the oxygen, as shown in Fig. 2. Theresult is consistent with the conclusions based on condensed-phase studies that oxygen atom transfer is favored by p-donors[45]. However, the reaction has not been observed previously foraromatic n-oxides. This work shows that oxygen atom transfer foraromatic n-oxides can occur with sufficiently strongp donors. Thedifferences in the electronic structures of 3PNO� and 4PNO� donot result in differences in reactivity with NO, although there aredifferences in the stabilities of the resulting products.
Acknowledgements
This paper is dedicated to Professor Veronica Bierbaum, inappreciation for all she has taught us about ion chemistry and
flowing afterglow kinetics. The work is supported by the NationalScience Foundation (CHE11-11777).
References
[1] M. Born, S. Ingemann, N.M.M. Nibbering, Reactivity of mono-halogen carbeneradical anions (CHX��; X = F, Cl and Br) and the corresponding carbanions(CH2X; X=Cl and Br) in the gas phase, J. Chem. Soc Perkin Trans. 2 (1996)2537–2547.
[2] J.J. Grabowski, S.J. Melly, Formation of carbene radical anions: gas-phasereaction of the atomic oxygen anion with organic neutrals, Int. J. MassSpectrom. Ion Processes 81 (1987) 147–164.
[3] R.N. McDonald, Generation, thermochemistry, and chemistry of carbene anionradicals and related species, Tetrahedron 45 (1989) 3993–4015.
[4] G.D. Sargent, C.M. Tatum Jr., S.M. Kastner, Carbene radical anions. New speciesof reactive intermediate, J. Amer. Chem. Soc. 94 (1972) 7174–7176.
[5] S.M. Villano, N. Eyet,W.C. Lineberger, V.M. Bierbaum, Gas-phase carbene radicalanions: new mechanistic insights, J. Am. Chem. Soc. 130 (2008) 7214–7215.
[6] S.M. Villano, N. Eyet, W.C. Lineberger, V.M. Bierbaum, Gas-phase reactions ofhalogenated radical carbene anionswith sulfur and oxygen containing species,Int. J. Mass Spectrom. 280 (2009) 12–18.
[7] R.N. McDonald, A.K. Chowdhury, Hypovalent radicals. 7. Gas-phase generationof phenylnitrene anion radical and its reactionwith phenyl azide, J. Am. Chem.Soc. 102 (1980) 5118–5119.
[8] R.N. McDonald, A.K. Chowdhury, D.W. Setser, Gas-phase generation ofphenylnitrene anion radical – proton affinity and DHf of PhN– and itsclustering with ROH molecules, J. Am. Chem. Soc. 103 (1981) 6599–6603.
[9] P.S. Drzaic, J.I. Brauman, A determination of the triplet-singlet splitting inphenylnitrene via photodetchment spectroscopy, J. Am. Chem. Soc. 106 (1984)3443–3446.
[10] P.S. Drzaic, J.I. Brauman, Electron photodetachment from phenylnitrene,anilide, and benzyl anions. Electron affinities of the anilino and benzyl radicalsand phenylnitrene, J. Phys. Chem. 88 (1984) 5285–5290.
[11] R.N. McDonald, S.J. Davidson, Electron photodetachment of the phenylnitreneanion radical: EA, DH�
f, and the singlet-triplet splitting for phenylnitrene,J. Am. Chem. Soc. 115 (1993) 10857–10862.
[12] M.J. Travers, D.C. Cowels, E.P. Clifford, G.B. Ellison, Photoelectron spectroscopyof phenylnitrene anion, J. Am. Chem. Soc. 114 (1992) 8699–8701.
[13] N.R. Wijeratne, M.D. Fonte, A. Ronenius, P.J. Wyss, D. Tahmassebi, P.G.Wenthold, Photoelectron spectroscopy of chloro-substituted phenylnitreneanions, J. Phys. Chem. A 113 (2009) 9467–9473.
[14] M.J. Travers, D.C. Cowles, E.P. Clifford, G.B. Ellison, P.C. Engelking, Photoelectronspectroscopy of the CH3N– ion, J. Chem. Phys. 111 (1999) 5349–5360.
[15] D.E. Herbranson, M.D. Hawley, Electrochemical reduction of p-nitrophenylazide: evidence consistent with the formation of p-nitrophenylnitrene anionradical as a short-lived intermediate, J. Org. Chem. 55 (1990) 4297–4303.
[16] S. Murata, R. Nakatsuji, H. Tomioka, Mechanistic studies of pyrene-sensitizeddecomposition of p-butylphenyl azide: generation of nitrene radical anionthrough a sensitizer-mediated electron transfer from amines to the azide,J. Chem. Soc. Perkin Trans. 2 (1995) 793–799.
[17] D.A. Van Galen, J.H. Barnes, M.D. Hawley, The electrochemical reduction offluorenone tosylhydrazone. Evidence consistent with the formation to thetosyl nitrene anion radical, J. Org. Chem. 51 (1986) 2544–2550.
[18] N.R. Wijeratne, P.G. Wenthold, Structure and reactivity of benzoyl nitreneradical anion in the gas phase, J. Org. Chem. 72 (2007) 9518–9522.
[19] N.R.Wijeratne, P.G.Wenthold, Benzoylnitrene radical anion: a new reagent forthe generation of M-2H anions, J. Am. Soc. Mass Spectrom. 18 (2007)2014–2016.
[20] N.R. Wijeratne, P.G. Wenthold, Thermochemical studies of benzoylnitreneradical anion: the N–H bond dissociation energy in benzamide in the gasphase, J. Phys. Chem. A 111 (2007) 10712–10716.
[21] Z.P. Demko, K.B. Sharpless, A click chemistry approach to tetrazoles byHuisgen1,3-dipolar cycloaddition: synthesis of 5-acyltetrazoles from azides and acylcyanides, Angew. Chem. Int. Ed. 41 (2002) 2113–2116.
[22] Z.P. Demko, K.B. Sharpless, A click chemistry approach to tetrazoles byHuisgen1,3-dipolar cycloaddition: synthesis of 5-sulfonyl tetrazoles from azides andsulfonyl cyanides, Angew. Chem. Int. Ed. 41 (2002) 2110–2113.
[23] H.C. Kolb, M.G. Finn, K.B. Sharpless, Click chemistry: diverse chemical functionfrom a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004–2021.
[24] C.W. Tornoe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase:[1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditionsof terminal alkynes to azides, J. Org. Chem. 67 (2002) 3057–3064.
[25] M. Al-Za'al, H.C. Miller, J.W. Farley, Observation of metastable states in theautodetachment continuum of the negative molecular ion amidogen ion(1�)(NH�), Chem. Phys. Lett. 131 (1986) 56–59.
[26] P.C. Engelking,W.C. Lineberger, Laser photoelectron spectrometry of imidogen(–) (NH�): electron affinity and intercombination energy difference inimidogen, J. Chem. Phys. 65 (1976) 4323–4324.
[27] J.W. Farley, High resolution laser spectroscopy ofmolecular ions, Proc. SPIE-Int.Soc. Opt. Eng. 1858 (1993) 92–100.
[28] K.K. Lykke, D.M. Murray, W.C. Neumark, High-resolution studies of autode-tachment in negative ions, Philos. Trans. R. Soc. Lond. A 324 (1988) 179–196.
[29] H.C. Miller, M. Al-Za'al, J.W. Farley, High-resolution measurement of theinfrared rotation–vibration spectrum of nitrogen hydride anion (NH1�),AIP Conference Procceedings 160 (1987) 354–355.
[(Fig._5)TD$FIG]
Fig. 5. Calculated distributions in 3PNO�, and 4PNO� and pyridine-n-oxide.Charges on hydrogen have been summed into heavy atoms.
74 D. Koirala et al. / International Journal of Mass Spectrometry 378 (2015) 69–75
168
[30] H.C. Miller, M. Al-Za'al, J.W. Farley, Measurement of hyperfine structure in theinfrared rotation–vibration spectrum of amidogen ion(1�) (NH�), Phys. Rev.Lett. 58 (1987) 2031–2034.
[31] D.M. Neumark, K.R. Lykke, T. Andersen,W.C. Lineberger, Infrared spectrum andautodetachment dynamics of NH, J. Chem. Phys. 83 (1985) 4364–4373.
[32] S. Srivastava, N. Sathyamurthy, Ab initio potential energy curves for the groundand low lying excited states of NH� and the effect of 2S� states onL-doublingof the ground state X2P, J. Phys. Chem. A 117 (2013) 8623–8631 (andreferences therein).
[33] U. Hoper, P. Botschwina, H. Koppel, Theoretical study of the Jahn–Teller effectin X�2E CH3O, J. Chem. Phys. 112 (2000) 4132–4142.
[34] D.L. Osborn, D.J. Leahy, D.M. Neumark, Photodissociation spectroscopy anddynamics of CH3O and CD3O, J. Phys. Chem. A 101 (1997) 6583–6592.
[35] P.J. Marinelli, J.A. Paulino, L.S. Sunderlin, P.G. Wenthold, J.C. Poutsma, R.R.Squires, A tandem selected ion flow tube-triple quadrupole instrument,Int. J. Mass Spectrom. Ion Processes 130 (1994) 89–105.
[36] T. Su,W.J. Chesnavich, Parametrization of the ion-polar molecule collision rateconstant by trajectory calculations, J. Chem. Phys. 76 (1982) 5183–5185.
[37] H. Sawanishi, K. Tajima, T. Tsuchiya, Studies on diazepines. XXVIII. Synthesesof 5H-1,3-diazepines and 2H-1,4-diazepines from 3-azidopyridines, Chem.Pharm. Bull. 35 (1987) 4101–4109.
[38] T. Itai, S. Kamiya, Potential anticancer agents. II. 4-Azidoquinoline and4-azidopyridine derivatives, Chem. Pharm. Bull. 9 (1961) 87–91.
[39] T. Itai, S. Kamiya, Potential anticancer agents. XI. Synthesis of 4- and5-azidopyridazine 1-oxide, Chem. Pharm. Bull. 11 (1963) 1059–1064.
[40] K.N. Crabtree, K.J. Hostetler, T.E. Munsch, P. Neuhaus, P.M. Lahti, W. Sander, J.S.Poole, Comparative study of the photochemistry of the azidopyridine 1-oxides,J. Org. Chem. 73 (2008) 3441–3451.
[41] Y. Shao, L.F. Molnar, Y. Jung, J. Kussmann, C. Ochsenfeld, S.T. Brown, A.T.B.Gilbert, L.V. Slipchenko, S.V. Levchenko, D.P. O’Neill, R.A. DiStasio, R.C. Lochan,T. Wang, G.J.O. Beran, N.A. Besley, J.M. Herbert, C.Y. Lin, T. Van Voorhis, S.H.Chien, A. Sodt, R.P. Steele, V.A. Rassolov, P.E. Maslen, P.P. Korambath, R.D.Adamson, B. Austin, J. Baker, E.F.C. Byrd, H. Dachsel, R.J. Doerksen, A. Dreuw, B.D. Dunietz, A.D. Dutoi, T.R. Furlani, S.R. Gwaltney, A. Heyden, S. Hirata, C.P. Hsu,G. Kedziora, R.Z. Khalliulin, P. Klunzinger, A.M. Lee, M.S. Lee, W. Liang, I. Lotan,
N. Nair, B. Peters, E.I. Proynov, P.A. Pieniazek, Y.M. Rhee, J. Ritchie, E. Rosta, C.D.Sherrill, A.C. Simmonett, J.E. Subotnik, H.L. Woodcock, W. Zhang, A.T. Bell, A.K.Chakraborty, D.M. Chipman, F.J. Keil, A. Warshel, W.J. Hehre, H.F. Schaefer, J.Kong, A.I. Krylov, P.M.W. Gill, M. Head-Gordon, Advances in methods andalgorithms in a modern quantum chemistry program package, Phys. Chem.Chem. Phys. 8 (2006) 3172–3191.
[42] S.A. Chacko, P.G. Wenthold, The negative ion chemistry of nitric oxide in thegas phase, Mass Spectrom. Rev. 25 (2006) 112–126.
[43] N.J. Rau, E.A. Welles, P.G. Wenthold, Anionic substituent control of theelectronic structure of aromatic nitrenes, J. Am. Chem. Soc. 135 (2013)683–690.
[44] H.Y. Afeefy, J.F. Liebman, S.E. Stein, Neutral thermochemical data, in: P.J.Linstrom, W.G. Mallard (Eds.), NIST Chemistry WebBook, NIST StandardReference Database Number 69, National Institute of Standards andTechnology, Gaithersburg, MD 20899, 2014 (retrieved 11.02.14).
[45] T. Yoshimura, K. Asada, S. Oae, Deoxygenation of tertiary amine oxides withcarbon disulfide, Bull. Chem. Soc. Jpn. 55 (1982) 3000–3003.
[46] M. Hamana, B. Umezawa, S. Nakashima, Tertiary amine oxides. XIV. Reactionsof N-(p-dimethylaminophenyl)nitrones, having pyridine, quinoline or its N-oxide, as a-substituents, with carbon disulfide, Chem. Pharm. Bull. (Tokyo) 10(1962) 969–974.
[47] J. Nakayama, A. Ishii, Chemistry of dithiiranes, 1,2-dithietanes, and1,2-dithietes, Adv. Heterocycl. Chem. 77 (2000) 221–284.
[48] M.F. Zipplies, M.J. De Vos, T.C. Bruice, The formation of thiiranes fromolefins inthe course of the deoxygenation of tertiary amine N-oxides by carbondisulfide, J. Org. Chem. 50 (1985) 3228–3230.
[49] V. Saheb, Quantum chemical and theoretical kinetics study of the O(3P) +CS2reaction, J. Phys. Chem. A 115 (2011) 4263–4269 (and references therein).
[50] J.W. Hudgens, J.T. Gleaves, J.D. McDonald, Infrared chemiluminescence studiesof the reactions of oxygen (3P) atoms with carbon disulfide and carbonmonosulfide, J. Chem. Phys. 64 (1976) 2528–2532.
[51] S.E. Barlow, V.M. Bierbaum, Reactions of O–+N2O at 300K: the totally labeledexperiments, J. Chem. Phys. 92 (1990) 3442–3447.
[52] C.H. DePuy, Fragmentation of organic anions induced by exothermic additionreactions, Org. Mass Spectrom. 20 (1985) 556–559.
D. Koirala et al. / International Journal of Mass Spectrometry 378 (2015) 69–75 75
169
