Fragmentation reactions of thiourea- and urea-compounds examined by tandem MS-, energy-resolved CID experiments, and theory

Article abstract of DOI:10.1016/j.ijms.2012.06.023

Fragmentation reactions of thiourea- and urea-compounds, which are promising reagents for chemical crosslinking (XL), are investigated in detail by collision-induced dissociation (CID) experiments in a quadrupole ion trap (QIT), energy-resolved CID experi

Full text of DOI:10.1016/j.ijms.2012.06.023

International Journal of Mass Spectrometry 330–332 (2012) 124–133
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International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Fragmentation reactions of thiourea- and urea-compounds examined by tandem
MS-, energy-resolved CID experiments, and theory
Francesco Falvoa, Lukas Fiebiga, Frank Dreiockera, Ran Wangb, P.B. Armentroutb, Mathias Schäfera,∗
a Department of Chemistry, Institute of Organic Chemistry, University of Cologne, Greinstraße 4, 50939 Köln, Germany
b Department of Chemistry, University of Utah, Salt Lake City, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2012
Received in revised form 26 June 2012
Accepted 26 June 2012
Available online 11 July 2012
Fragmentation reactions of thiourea- and urea-compounds, which are promising reagents for chemi-
cal crosslinking (XL), are investigated in detail by collision-induced dissociation (CID) experiments in a
quadrupole ion trap (QIT), energy-resolved CID experiments, and computational modeling. For this study,
an array of six labeled and unsymmetrical substituted thiourea- and urea-derivatives were synthesized,
which allow unambiguous characterization of competing fragmentation pathways. The results of the
QIT-CID-experiments are explored in detail for two compounds and confirmed by results for the other
four. These results document the subtle competition of characteristic fragmentation pathways of this
class of compounds. The multi dimensional investigations of the characteristic fragmentation reactions
allow reliable structure proposals of prominent product ions. Energy-dependent CID experiments on two
of the six compounds lead to breakdown curves that show similar relative threshold energies for the for-
mation of the product ions on which the functioning of the XL application relies. The experimental results
are in full consistency with the results from in-depth computations. For the dominant fragmentations
observed, the transition states for moving the proton from the most basic site of the precursor ion (the
thiourea-sulfur or the urea carbonyl oxygen) to less basic heteroatoms in the protonated molecular ion is
the necessary and decisive step that determines the extent of the charge-driven fragmentation processes
that follow.
Keywords:
Collision induced dissociation
Energy-dependent dissociation
Breakdown curve
Reaction mechanism
Computational modeling
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
them effective CID-labile XL-reagents, are the fragmentation of the
urea-moiety and the cleavage of an amide bond adjacent to the
Chemical cross linking (XL) is an established analytical strategy
to study three-dimensional protein structures and to map pro-
tein interfaces [1–4]. However, the unambiguous, sensitive, and
rapid identification of chemically modified amino acids in com-
plex peptide mixtures after enzymatic digestion is challenging as
only a relatively low amount of cross-linked products are present
along with a large majority of unmodified peptides. Tailor-made
cross linkers that fragment readily upon collisional activation offer
a solution for this problem as they give rise to characteristic frag-
ment ions or show characteristic neutral losses that in turn afford
facile and reliable tandem mass spectrometric analysis [5–11].
Recently, we have introduced urea- and thiourea-compounds for
XL that are especially suited for collision-induced dissociation (CID)
analysis (see compounds 7a and 7b in Scheme 1) [5–7,11]. Their
typical and highly preferred fragmentation reactions, which make
thiourea-moiety [6,11]. These different competing fragmentation
reactions, their mechanisms, and the structure of their key product
ions are examined here in detail [12,13].
The rather new application as efficient XL-reagents was adapted
from the well documented nucleophilic reactivity of the thiourea
sulfur of N-terminal phenyl thiocarbamoyl compounds, which is
essential for the Edman degradation reaction [5,14] and also the
basis for ladder sequencing of peptides by MS/MS [15,16]. In addi-
tion, urea- and thiourea-compounds have attracted attention as
potent organocatalysts because of their effective influence on reac-
tions via their unique hydrogen-bonding patterns [17–22]. Finally,
the urea-moiety is frequently found in pseudopeptides, which are
interesting new biodegradable polymeric materials for biomedical
applications [23]. Most importantly, urea is the main nitrogen-
containing substance in the urine of mammals as it is the major
end product of nitrogen metabolism and as such a ubiquitous com-
pound of biochemical importance [24].
In this contribution, we turn our attention to the fundamentals
of the characteristic CID pathways of protonated and sodiated alkyl
∗
Corresponding author. Tel.: +49 0 2214703086; fax: +49 0 2214703064.
E-mail address: mathias.schaefer@uni-koeln.de (M. Schäfer).
1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijms.2012.06.023

F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
125
thiocarbamoyl and alkyl carbamoyl molecular ions to ultimately
2. Materials and methods
understand and evaluate the obvious differences in reactivity of
urea and thiourea vs. simple amide moieties in the gas phase. To
accomplish this complex analytical task, we first synthesized a set
of unsymmetrical thiourea- and urea-compounds, 1–6 shown in
Scheme 1, which are simplified analogs of the reagents used for
XL, 7a and 7b. These compounds allow unambiguous identification
of the competing fragmentation reactions on the basis of charac-
teristic product ions generated in quadrupole ion traps (QIT) MSn
product ion experiments. As illustrated in Scheme 1, compounds
1 and 2 are deuterium labeled thiocarbamoyl- and carbamoyl-
derivatives that come with the key structural features also present
in the XL-reagents, i.e., an alkyl thiourea/urea moiety connected
to butyric acid amides (see compounds 7a and 7b in Scheme 1)
[6,11]. As this pair of derivatives shows only a minimal structural
difference, the discussion of the analytical results is focused on
them, but results for compounds 3–6 are considered as well. The
structures of compounds 3–6 differ more substantially as they are
decorated with diverse alkyl groups (propyl and cyclohexyl) and
even with a glycylmethylester-moiety (compound 4). These com-
pounds are included in the study to probe if subtle differences in
inductive and steric effects, which can be expected from the pres-
ence of differing alkyl groups (R and Rꢀ), have an influence on the
fragmentation pathways. These results allow further evaluation of
the experimental findings for compounds 1 and 2.
2.1. Mass spectrometry
All chemicals and solvents were used as purchased without
further purification (Acros Organics, Aldrich, Fluka, Merck and Lan-
caster). Tandem-MS CID-experiments with monoisotopic precursor
ion selection (conducted with the magnetic (B) and electric (E) sec-
tors, i.e., the BE part) were carried out in the quadrupole ion trap
(QIT)ofahybridBE-QIT Finnigan MAT 900 massspectrometer (Ther-
moFisher Scientific, Bremen). Exact ion masses of all protonated and
sodiated precursor ions as well as of the MSn product ions were
measured in the orbitrap analyzer of a LTQ Orbitrap XL instrument
at resolutions >30,000 FWHH (ThermoFisher Scientific, Bremen). The
theoretical masses of all ions discussed match the exact ion masses
determined (external calibration: ꢀm ≤ 3 ppm; internal standards:
≤1 ppm).
Energy-resolved MS2 experiments were conducted on a Bruker
Esquire 3000 plus ion trap mass spectrometer (Bruker Dalton-
ics GmbH, Bremen, Germany) equipped with an electrospray
ionization (ESI) source yielding the breakdown curves shown
below. 50 MS2 product ion spectra were acquired at every acti-
vation voltage and the ion abundances were averaged to yield
the respective data points presented below. Nitrogen was used
as the drying and nebulizer gas at a temperature of 295 ◦C, a
flow rate of 5 l/min, and a back-pressure of 10 psi. The needle
voltage was always set to 4 kV. Protonated precursor ions of com-
pounds 1 and 2 were generated in the gas phase by introducing
a 10−6 M methanolic solution of the analyte with 0.1 vol% tri-
fluoroacetic acid into the ESI source at a flow rate of 9 ␮l/min.
Breakdown curves were determined by monoisotopic mass selec-
tion of the corresponding parent ion and applying an activation
voltage for a period of 20 ms. Helium was used as the colli-
sion gas. Activation voltages then were increased in a stepwise
The fragmentation behavior of protonated and sodiated molecu-
lar ions of the model compounds 1–6 was examined by electrospray
ionization (ESI)-MS/MS in a QIT. The energetics of the dissocia-
tion reactions of compounds 1 and 2 are subsequently probed
by energy-resolved QIT-CID experiments and by computational
modeling [25–30]. The energy-dependent CID experiments yield
breakdown curves that allow the approximate determination of
relative appearance energies of product ions, which are then com-
pared to predictions by theory [25,26,29,30].
O
S
H
N
H
D3C
D3C
N
N
H
N
H
CH3
N
H
N
H
CH3
O
O
1
2
4
S
S
O
H
N
H
N
CH3
N
H
N
H
CH3
N
N
H
O
H
O
O
3
S
O
H
H
N
N
N
H
N
H
N
H
N
H
O
O
6
5
X
H
H
N
N
R
N
H
N
R'
H
O
O
7a, X = S
7b, X = O
Scheme 1. Alkyl thiourea and alkyl urea compounds 1–6 examined in this work. Symmetric CID-labile XL-reagents 7a (X S) and 7b (X O) are presented here for structural
comparison. XL-reagents 7a and 7b, are initially activated amine sensitive N-hydroxy succinimide (NHS) ester, which are shown here after condensation reaction with peptide
amine moieties (R and Rꢀ).

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F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
fashion until complete dissociation of the ion of interest was
observed.
2.4. Synthesis of N-methyl-4-(3-propyl-thioureido)-butyramide
(3)
The synthesis of compound 3 was carried out by the reaction
of the corresponding isothiocyanate (see synthesis of compound 1)
with propylamine.
2.2. Synthesis of
N-methyl-4-(3-trideuteromethyl-thioureido)-butyramide (1)
Tert-Butyloxycarbonyl (BOC) protection of the amine func-
tionality of 4-aminobutyric acid (5 g; 48.5 mmol) was achieved
by reaction with di-tert-butyl dicarbonate (22.21 g; 101.8 mmol)
and triethylamine (29.45 g; 291 mmol) in methanol in 10 h [31].
After the evaporation of the solvent, the residual oil was dis-
solved in 150 ml saturated sodium hydrogen carbonate solution
and extracted with ethylacetate. The aqueous phase was acidified
with 1 N HCl to pH 2–3 and also extracted with ethylacetate. The
combined organic layers were dried over magnesium sulfate and
finally a white solid was obtained.
A mixture of the BOC-protected amino acid (2.3 g; 10 mmol),
methylamine hydrochloride (0.81 g; 12 mmol), and triethylamine
(3.54 g; 35 mmol) in 200 ml dichloromethane was cooled to 0 ◦C.
Then a 1-propanephosphonic acid cyclic anhydride (T3P) solution
in dimethyl formamide (DMF, 50%, w/w) was added drop wise and
the mixture was allowed to warm to room temperature overnight
[32]. The mixture was washed with 1 N HCl, 1 N NaOH, satu-
rated sodium chloride solution and dried over magnesium sulfate.
After evaporation of the solvent, tert-butyl 4-(methylamino)-4-
oxobutylcarbamate was isolated as a colorless solid.
2.5. Synthesis of [4-(3-propyl-thioureido)-butyrylamino]-acetic
acid methyl ester (4)
A mixture of 5 g BOC-protected 4-aminobutyric acid (25 mmol),
3.61 g 4–dimethylaminopyridine (DMAP; 30 mmol), 4.3 g glycine
methylester hydrochloride (34 mmol), and 4.34 g triethylamine
(43 mmol) in dichloromethane was cooled to 0 ◦C. 7.11 g dicyclo-
hexyl carbodiimide (DCC; 34 mmol) were added after 20 min and
the mixture was warmed up to room temperature over night. The
mixture was washed with 1 N HCl, 1 N NaOH, saturated sodium
chloride solution and dried over magnesium sulfate. After evapora-
tion of the solvent, 5.6 g (82%) of the crude product were purified by
column chromatography with dichloromethane/methanol (10:1)
as a white solid.
Cleavage of the BOC-group occurs in trifluoroacetic acid within
1 h at room temperature. The resulting amine-salt was converted
to the respective isothiocyanate as described above. The reac-
tion of 0.916 g isothiocyanate (4.24 mmol) with 0.5 g propylamine
(8.47 mmol) leads to thiourea-derivative 4 (0.962 g; 83%).
The BOC-deprotection (1.2 g; 5.6 mmol) was carried out in tri-
fluoroacetic acid within 1 h at room temperature. The solution was
evaporated to dryness in vacuo and the residue triturated with
dry diethyl ether. The residual oil was disintegrated under fresh
diethylether, filtered and dried in vacuo. The resulting colorless oil
was used without purification.
2.6. Synthesis of
N-cyclohexyl-4-(3-propyl-thioureido)-butyramide (5)
Compound 5 (0.015 g; 59%) was synthesized in analogy to com-
pound 3.
For the synthesis of the isothiocyanate, 1.19 g of the amine
(5 mmol) were dissolved in a mixture of acetonitrile and carbon
disulfide (0.4 g; 5.25 mmol). After slowly adding 1.21 g of triethy-
lamine (11.5 mmol), the reaction mixture was stirred for 20 min
before methyl chloroformate (0.57 g; 5.6 mmol) was added. After
refluxing for 3 h, the mixture was cooled down to room temper-
ature and poured into water [33]. The organic layer was washed
with diluted hydrochloric acid and brine, dried over magnesium
sulfate and concentrated under reduced pressure. The resulting
yellowish oil was purified by column chromatography with ethyl
acetate/cyclohexane (2:1) and 0.61 g of the product was isolated as
colorless oil (73%).
For the reaction of the isothiocyanate with trideutero methy-
lamine hydrochloride (0.07 g; 1 mmol), the amine was suspended
in 15 ml acetonitrile and treated with 0.11 g triethylamine
(1.05 mmol). After 15 min, 0.16 g of the isothiocyanate (1 mmol)
were added at 0 ◦C and the reaction mixture was brought to
20 ◦C within 20 h. After the evaporation of the solvent, the
crude product was purified by column chromatography with
dichloromethane/methanol (10:1). The methyl thiocarbamoyl-
derivative 1 was isolated as a colorless solid (0.17 g; 88%).
2.7. Synthesis of N-cyclohexyl-4-(3-propyl-ureido)-butyramide
(6)
Compound 6 (0.066 g; 98%) was synthesized in analogy to com-
pound 2.
2.8. Computations
To find the global energy minimum and all low-energy geome-
tries, a large number of possible conformations were screened using
a simulated annealing methodology with the AMBER program and
the AMBER force field based on molecular mechanics [35]. All pos-
sible structures identified this way were subsequently optimized
using NWChem [36] at the HF/3-21G level [37,38]. Unique struc-
tures for each system were optimized using Gaussian 09 [39] at
the B3LYP/6-31G(d,p) level of theory [40,41] with the “loose” key-
word (maximum step size 0.01 au and an RMS force of 0.0017 au)
to facilitate convergence. A series of relaxed potential energy sur-
face (PES) scans at the B3LYP/6-31+G(d) level were performed to
locate transition state and intermediate structures occurring along
the potential energy surfaces for fragmentation. Unique struc-
tures of all stable conformations; intermediates; transition states;
and products obtained from these procedures were then chosen
for higher-level geometry optimization and frequency calculations
using the B3LYP/6-311+G(d,p) level of theory [42]. Each transition
state was found to contain one imaginary frequency and each inter-
mediate was vibrationally stable. Vibrational frequencies were also
calculated at this level and were scaled by 0.99 [43] in determin-
ing zero-point vibrational energy (ZPE) corrections. Single-point
energies were determined at the B3LYP; B3P86; and MP2(full) lev-
els using the 6-311+G(2d,2p) basis set and the B3LYP/6-311+G(d,p)
geometries.
2.3. Synthesis of
N-methyl-4-(3-trideuteromethyl-ureido)-butyramide (2)
Compound 1 was dissolved in an aqueous sodium hydrox-
ide solution. After 10 min a 35% hydrogen peroxide solution was
added drop wise at 0 ◦C to the reaction mixture and the mix-
ture was allowed to warm to room temperature over night [34].
After evaporation to dryness the raw product was purified by
column chromatography with dichloromethane/methanol (5:1).
Compound 2 was isolated as a colorless solid (0.05 g; 68%).

F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
127
159
100
90
80
70
60
50
40
30
20
10
+
m 34u
SH
- CD3NH2
H
N
D3C
N
H
N
H
CH3
O
[1 + H]+
m/z 193
[1 + H]+
m/z 193
m 31u
- CH3NH2
162
117
100
86
0
60
70
80
90
100 110 120 130 140 150 160 170 180 190 200
m/z
Fig. 1. (+)ESI-QIT-MS2 product ion spectrum of the protonated precursor ion [1+H]+ at m/z 193. The precursor ion is completely depleted by CID.
3. Results and discussion
A third charge-driven fragmentation reaction, i.e., the loss of
trideutero methyl isothiocyanate CD3NCS (ꢀm 76 u) is found in
the MS2-spectrum of [1+H]+ (see Scheme 2 and Fig. 1), the analog
of which (loss of 101 u) is also found for [5+H]+ (see SI: Scheme 1S
and Fig. 6S). The former reaction leads to the protonated 4-amino-
N-methyl-butyramide ion at m/z 117, which can further fragment
to yield ions at m/z 110 and 86 upon loss of ammonia and methy-
lamine, respectively (see also SI: Fig. 1S). The m/z 100 product
could also be formed directly from [1+H]+ by loss of CD3NHCSNH2
(methyl thiourea, ꢀm 93 u) induced by nucleophilic attack of the
oxygen at the urea nitrogen, Scheme 2. Computations suggest that
the most likely pathway for formation of m/z 86 is by loss of CD3NCS
from the ␥-butyrolactame structure of m/z 162.
To understand the occurrence of QIT secondary fragmentation
processes, it is appropriate to recall the basics of ion activation and
fragmentation in QIT-MS in which only a selected precursor ion is
in resonance with the radiofrequency applied for activation. This
interaction increases the kinetic energy of the precursor ion and
the multiple collisions with the He-buffer gas lead to accumulation
of internal energy via the slow heating mechanism of activation
and eventually to CID [50]. All product ions generated by primary
fragmentation of the precursor ion are then rapidly cooled by dis-
sipative low energy collisions with the He background gas [51–53].
This is certainly true for low collision energies, whereas high colli-
sion energies lead to “hot” fragment ions that can dissociate further
before being effectively cooled by the helium in the trap [54,55]. In
Fig. 1 as well as in the other QIT-MS2 product ion spectra shown
in this contribution, the precursor ions are completely depleted
and therefore increased collision energies were applied. Hence,
in addition to primary fragmentation of the activated precursor
ion, secondary fragmentation reactions of product ions can also
occur, as frequently found at sufficiently high activation energies
deposited into resonantly excited precursor ions.
3.1. (+)ESI-QIT-MS2-experiments
3.1.1. Protonated molecular ion of compound 1: [1+H]+
The major fragmentation pathway of the protonated molecu-
lar ion of compound 1 at m/z 193 is the cleavage of the thiourea
moiety, i.e., loss of trideutero methylamine CD3NH2 (ꢀm 34 u) to
generate the product ion at m/z 159 (see Fig. 1). Additionally, the
loss of methylamine CH3NH2 (ꢀm 31 u) leading to formation of
a product ion at m/z 162 is found in the MS2-spectrum of model
compound 1 with moderate abundance (Fig. 1). In both cases, plau-
sible mechanisms include backside nucleophilic attack either of
the carbonyl oxygen onto the thiocarbonyl or of the thiourea car-
bonyl sulfur onto the amide carbonyl group, respectively, leading
to 7-membered heterocyclic products (Scheme 2). Alternatively,
simple loss of CD3NH2 would form a product ion stabilized by a
hydrogen bond between the thioisocyanate nitrogen and the amide
oxygen (m/z 159), as Scheme 2 illustrates. Loss of CH3NH2 can
lead to at least three different product ion structures found at m/z
162: (a) the 7-membered heterocyclic product ion, (b) a proto-
nated ␥-butyrolactame-derivative formed by nucleophilic attack of
the thiourea nitrogen onto the amide carbonyl carbon, and finally
(c) a rather unstable acylium ion, Scheme 2. These suggestions
are derived from the broadly accepted understanding of peptide
backbone amide bond fragmentation reaction mechanisms, which
were elucidated by MS/MS experiments and in-depth computa-
tions [44–47]. In particular protonated oxazolone b-ions formed
by nucleophilic attack of adjacent carbonyl oxygens and cleavage
of an amide bond are reliably confirmed and explain the observed
stability of peptide b-ions [48]. However, acylium b-ions, which
were hypothesized at early stages of the research, have been sub-
sequently clearly disproved as such ions are very unstable because
of the facile exothermic loss of carbon monoxide, which converts
them into much more stable immonium a-ions [49]. Even though
the product ion at m/z 162 is not derived from a protonated ␣-
amino acid and therefore a stable immonium a-ion analog cannot
be formed by CO loss (a product not found in the MS2 spectrum),
we favor a 7-membered ring over an acylium ion structure for m/z
162 (see Scheme 2). Although nucleophilic attack of the thiourea
sulfur cleaving off the neighboring amide bond is the characteristic
and predominant reaction of the thiourea-XL reagent (compound
7a in Scheme 1), this fragmentation pathway is outmatched by the
cleavage of the thiourea-moiety in protonated molecular ions of
compound 1 (see Fig. 1 and Scheme 2) [6].
All charge-driven fragmentation reactions shown in Scheme 2
rely on the ionizing proton to be located adjacent to the cova-
lent bond that is to be cleaved. The distribution of the proton over
various sites of different basicity in the molecular ion that is the pre-
condition for all of the subsequent cleavages can be understood by
the mobile proton model [44,46,47,56–63]. According to this con-
cept, the proton migrates upon collisional activation (CA) from the
most basic site in the precursor ion, in this case the thiourea-sulfur,
to the decisive location for each of the discussed charge-driven
fragmentation reactions, as indicated in Scheme 2. (In this context
and throughout the remainder of the paper, we use the term CA to
indicate activated steps that occur before dissociation.)

128
F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
+
SH
[1 + H]+
H
N
D3C
N
H
N
H
CH3
D3C
m/z193
O
S
Collision
H
N
S
+
D3C
Activation
H
N
N
N
H
CH3
D3C
+
H2
N
H
N
CH3
O
H2
O
S
H2
N
+
O
m 34u
+
N
N
H
CH3
m 76u
H
NH
- CD3NH2
O
- CD3NCS
m/z100
m 31u
- CH3NH2
H
H
N
+
N
+
H3C
H
H
H3N
CCH3
N
+
O
N
O
NH
D3C
m/z117
S
S
m 31u
S
O
or
- CH3NH2
+
D3C
O
S
N
N
or
H
H
C
S
H
+
O
H
N
N
m 76u
+
D3C
N
N
OH
- CD3NCS
N
H
H
H
+
O
m/z 86
m/z159
m/z 162
Scheme 2. Charge-driven fragmentation pathways observed in the (+)ESI-QIT-MS2 product ion spectrum of the protonated molecular ion [1+H]+ of 1 at m/z 193 (see Fig. 1).
3.1.2. Sodiated molecular ion of compound 1: [1+Na]+
SH
S
In analogy with protonated 1, sodiated 1 also loses 34 u predom-
inantly and 76 u in lower abundance upon collisional activation.
However, exact ion mass determination of the sodiated precursor
ion [1+Na]+ at m/z 215 and the product ion (m/z 181) proves that
the respective mass shift of 34 u originates from the loss of hydro-
gen sulfide and not of CD3NH2 (compare Fig. 1 and Scheme 2).
To rationalize the loss of H2S and the formation of the sodiated
carbodiimide ion at m/z 181 starting from the sodiated thiourea-
compound 1, we assume a preceding tautomerization upon CA that
converts the predominant thione to the respective enthiol tautomer,
which then eventually cleaves off H2S in a subsequent fragmenta-
tion reaction (Scheme 3) [64]. The elimination of hydrogen sulfide
from the sodiated precursor ion evidences the importance of the
ionizing proton for the loss of CD3NH2, which is absent in this case
(SI: Scheme 2S). However, the loss of trideutero methyl isothio-
cyanate CD3NCS depends on a hydrogen shift to form the sodiated
4-amino-N-methyl-butyramide at m/z 139. The loss of H2S is found
in all QIT-MS2 product ion spectra of sodiated thiourea-compound
R
R'
R
R'
N
N
H
N
H
N
H
Scheme 3. Thione-enthiol tautomerization of thiourea-compounds. In solution and
in the gas phase, the equilibrium lies predominantly on the left side at the thione
[64].
precursor ions (i.e., compounds 3–5; see SI: Figs. 2S, 5S, 7S and
Scheme 2S), which was additionally confirmed by H/D exchange
experiments with compound 5 (loss of ꢀm 36 u = D2S for CID of the
triply deuterated [D35 + Na]+ molecular ion; SI: Fig. 10S) (see Fig. 2).
3.1.3. Protonated molecular ion of compound 2: [2+H]+
In Fig. 3, the ESI-QIT-MS2 product ion spectrum of the proto-
nated molecular ion of 2 is presented. Only the loss of CD3NH2
and not that of CH3NH2 is observed (see also Scheme 4). In lower
CD3
+ Na+
N
H
N
181
100
90
80
70
60
50
40
30
20
10
0
C
N
CH3
O
m 34u
- H2S
+ Na+
H
N
H2N
CH3
[1 + Na]+
m/z 215
O
m 76 u
139
- CD3NCS
70
80
90 100 110 120 130 140 150 160 170 180 190 200 210 220
m/z
Fig. 2. ESI-QIT-MS2-product ion spectrum of the sodiated precursor ion [1+Na]+ at m/z 215. The precursor ion is completely depleted by CID.

F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
129
143
100
90
80
70
60
50
40
30
20
10
0
+
OH
H
D3C
N
N
H
N
H
CH3
O
[2 + H]+
m/z 177
m 34 u
- CD3NH2
[2 + H]+
m/z 177
117
100
100
60
70
80
90
110 120
130 140
150 160
170 180
190
200
m/z
Fig. 3. ESI-QIT-MS2 product ion spectrum of the protonated precursor ion [2+H]+ at m/z 177. The precursor ion is completely depleted by CID.
abundance, the trideutero methyl isocyanate is cleaved off to form
the ion at m/z 117. The minor product ion at m/z 100 can again be
formed by ammonia loss from m/z 117 or directly from the parent
ion by loss of methyl urea (CD3NHCONH2), as Scheme 4 illustrates.
Notably, the m/z 86 product found in Fig. 1 is not observed here,
which suggests that this product is not formed from m/z 117, but
rather from m/z 162 formed by initial CH3NH2 loss, the analog of
which is not formed from [2+H]+. To rationalize the absence of the
loss of CH3NH2 in the fragmentation pattern of the urea compound
2, the substantially higher electronegativity of oxygen compared
to that of sulfur offers a solution. On this basis, it is reasonable
to assume that the ionizing proton is more loosely bound to the
thiourea sulfur in the protonated molecular ion of compound 1
than in the urea [2+H]+ ion. Consequently, its migration upon CA
to less basic sites in the molecule is easier in [1+H]+. In the proto-
nated molecular ion [2+H]+, the ionizing proton is only transferred
to the nitrogens of the urea moiety but not to the amide nitro-
gen, presumably because of the lower gas-phase basicity of the
latter [44,58], such that the loss of CH3NH2 is not observed. In
addition, the absence of a product ion at m/z 146 formed by the
loss of CH3NH2 in the QIT-MS2 spectrum of [2+H]+ (see Fig. 3) sup-
ports the assumption that an acylium ion structure is unlikely for
the respective product ion at m/z 162 in Fig. 1. This is because the
formation of the latter should be largely unaffected by the presence
of either an urea or a thiourea moiety in the molecule, whereas this
substitution should influence formation of the cyclic products.
3.1.4. Sodiated molecular ion of compound 2: [2+Na]+
In Fig. 4, the product ions of the sodiated molecular ion [2+Na]+
of compound 2 are presented. The dominant fragmentation chan-
nels in the MS2 product ion experiment are related to the formation
of alkyl isocyanates, which are either expelled as a neutral (loss of
60 u: CD3NCO, product ion m/z 139) or remain visible as a sodiated
product ion at m/z 165 (loss of CD3NH2). The latter fragmentation
reaction is remarkable because the loss of trideutero methylamine
CD3NH2 (ꢀm 34 u) is strongly preferred over the loss of water
(m/z 181) and the formation of a respective carbodiimide. This
striking difference in the fragmentation behavior of the sodiated
molecular ions of 1 and 2 can be related to differences in the keto-
enol tautomeric equilibrium of the urea- and thiourea-moiety (see
Scheme 3). Theoretical and experimental results show that the for-
mation of the enol form is more favored for thiourea-compounds
than for respective urea-compounds [64]. On this basis, it seems
reasonable to assume that the facilitated loss of hydrogen sulfide
observed for [1+Na]+, which relies on the proton shift from nitro-
gen to sulfur, is easier than the analogous loss of water from the
+
OH
H
[2 + H]+
D3C
N
N
H
N
H
CH3
m/z 177
O
Collision
Activation
O
O
H
N
D
+
H
N
N
H
CH3
D3C
N
+
H2
N
H
N
CH
3
O
H2
O
m 77 u
O
m 60 u
m 34 u
- CD3NCO
D3C
- CD3NH2
N
NH2
H
H
N
+
H3N
CH3
O
H
+
N
O
NH
O
+
H3CC
C
m/z 117
+
H
N
O
O
m/z 100
N
H
or
N
H
O
m/z 143
Scheme 4. Charge-driven fragmentation pathways observed in the (+)ESI-QIT-MS2 product ion spectrum of the protonated molecular ion [2+H]+ at m/z 177 (see Fig. 3 above).

130
F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
+ Na+
O
H
N
C
165
N
CH3
100
90
80
70
60
50
40
30
20
10
0
O
m 34u
+ Na+
139
- CD3NH2
H
N
H2N
CH3
[2 + Na]+
m/z 199
O
m 60 u
- CD3NCO
m 18 u
- H2O
181
60
70
80
90
100 110 120 130 140 150 160 170 180 190 200 210
m/z
Fig. 4. ESI-QIT-MS2-product ion spectrum of the sodiated precursor ion [2+Na]+ at m/z 199. The precursor ion is completely depleted by CID.
sodiated urea compound 2. In addition, the thermochemistry of
the H2O loss from urea-compounds vs. the H2S loss from thiourea-
derivatives strongly favors the former as the C O bond is much
more stable than the C S bond according to the assumptions of the
classical “double bond rule” [65]. Thus, the respective product ion
generated by loss of water is hardly visible in Fig. 4 (m/z 181).
Finally, the higher stability of isocyanates over isothiocyanates
reflected in their tautomeric equilibria with corresponding
cyanates and thiocyanates, respectively, might also have an influ-
ence on the fragmentation patterns of the sodiated molecular ions
of 1 and 2 as the formation of alkyl isocyanates is clearly the favored
option for compound 2 [66]. These considerations for compound
2 also hold true for the urea-compound 6, which delivers similar
results in MS2 product ion experiments (see SI: Figs. 11S–13S).
compromised by the experimental uncertainties and provide useful
energetic information [30].
In the present work, threshold activation voltages are deter-
mined with mass-selected precursor ions by gradually increasing
the activation voltage until complete depletion of the precursor
ion is observed. In doing so, a breakdown curve with a sigmoidal
shape is obtained from which the threshold voltage can either be
determined as the voltage at which the fragment ion intensity has
reached a certain percentage of the total ion intensity (e.g., 5% [30],
10% [29], or 50% [28]) or be determined by linear extrapolation
of the slope of the breakdown curve to the baseline [25]. The lat-
ter procedure is utilized here, where the data analyzed include the
most linear region of the rising curve.
Energy-dependent dissociation studies of compounds 1 and 2
yielded the breakdown curves shown in Figs. 5 and 6. Linear extrap-
olation of the product ion curves of interest, i.e., product ions at m/z
159 and m/z 162 in Fig. 5 and of product ion at m/z 143 in Fig. 6,
lead to nearly equal threshold voltages of 0.11–0.12 V. We estimate
an experimental error of at least 10% by judging the quality of the
linear fit and the deviations of ion abundances measured. However,
3.2. Energy-resolved QIT-MS2 product ion experiments of [1+H]+
and [2+H]+
Ion activation and CID in QIT-MS is a complex process as it relies
on multiple collisions with the He-buffer gas and hence it is difficult
to extract quantitative information on the collision energy depen-
dence for selected fragmentation reactions [52]. However, Schröder
and others have developed methods to convert phenomenological
appearance energies of product ions extracted from experimental
QIT-MS breakdown curves into actual critical energies of frag-
mentation reactions [25,26,30,67,68]. In the case of the spherical
QIT-MS Esquire 3000 plus by Bruker Daltonics in which the MS2
product ion experiments were conducted, the amplitude of the RF-
voltage used for excitation is controlled experimentally. This rather
abstract value can be transformed to estimated threshold ener-
gies of certain fragmentation reactions in relation to well described
fragmentation reactions of so called thermometer ions with well
documented threshold energies, e.g., fragmentation reactions of
substituted benzylpyridinium ions as suggested by DePauw and
others [67,69–73]. However, it must be stressed that the energy-
resolved product ion experiments in QIT-MS yield only rough
estimates as the CID process and the internal energy distribution
of the precursor ion is ill defined [25]. Threshold CID experiments
on guided-ion-beam-instruments with much better control of the
experimental conditions defining the source and individual colli-
sion events combined with accurate data evaluation [74–76] are
underway to determine correct absolute threshold energies for
the fragmentation reactions of interest here. Even though absolute
critical energies deduced from QIT-MS are unreliable, relative QIT-
MS threshold values of competing fragmentation reactions are less
100
80
m/z 193
m/z 100
m/z 117
m/z 159
m/z 162
60
40
20
0
linearextrapolation
linear extrapolation
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Ampl [V]
Fig. 5. Energy resolved (+)ESI-QIT-MS2 product ion experiments of the protonated
molecular ion [1+H]+ of compound 1 (precursor ion m/z 193). (Linear regression
analyses using data points from 0.12 to 0.21 V: m/z 159: y = 667.3·x − 77.2; R2 = 0.99;
m/z 162: y = 186.1·x − 21.9; R2 = 0.94.)

F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
131
100
80
60
40
20
0
linear extrapolation
100 u
117 u
143 u
177 u
0,00
0,05
0,10
0,15
0,20
0,25
0,30
Ampl [V]
Scheme 5. Computed reaction pathway of the migration of the ionizing proton from
the most basic site in the molecular ion [1+H]+ upon CA to the adjacent thiourea
nitrogen followed by dissociation to products at m/z 159. Dashed lines indicate
bonds being broken or formed. Only key steps are shown.
Fig. 6. Energy resolved (+)ESI-QIT-MS2 product ion experiments of the protonated
molecular ion [2+H]+ of compound 2 (precursor ion m/z 177). (Linear regres-
sion analysis using data points from 0.11 to 0.18 V: m/z 143: y = 1176.9·x − 128.7;
R
2 = 0.98.)
CID behavior of the protonated molecular ions of 1 and 2 were
closely examined by theory. Careful scans of the PES yield char-
acteristic transition states for loss of CD3NH2, Schemes 5 and 6.
In both schemes, the first step takes the ground state conformation
and rotates the (thio) urea group around a CN bond to form a higher
energy conformer of the protonated molecular ions. Then the pro-
ton is transferred from the S or O in the (thio) urea group to the
nitrogen adjacent to the labeled methyl group. The N-protonated
intermediates formed can then lose CD3NH2 readily by simple bond
cleavage to yield the protonated isothiocyanate structure of prod-
uct ion m/z 159 of [1+H]+ and the respective isocyanate structure
for the ion at m/z 143 of [2+H]+. At higher energies, nucleophilic
attack of the carbonyl oxygen at the urea carbon can form the
seven-membered ring forms of the two product ions. These hete-
rocyclic structures are higher in energy by 49–63 kJ/mol for [1+H]+
and 14–24 kJ/mol for [2+H]+ and therefore less likely to be formed.
Indeed, the energy of the heterocyclic product greatly exceeds that
of the rate-limiting TS in the thiourea case, whereas it is energeti-
cally accessible for the urea case.
the similar appearance energies of all three product ions lead to the
following hypotheses:
(1) As strongly different product ion yields of the competing frag-
mentation reactions are found (see Fig. 1), the two reaction
channels (i.e., the loss of CD3NH2 vs. CH3NH2 leading to the for-
mation of product ions at m/z 159 and m/z 162; Fig. 1) proceed
via different mechanisms including different entropic demands
of the transition state (TS), which in turn suggests different
product ion structures. For the [1+H]+ ion, this assumption
strongly points toward the formation of one of the heterocyclic
product ions at m/z 162 found as result of the nucleophilic
attack of the thiourea-sulfur or urea nitrogen onto the adjacent
amide bond and the protonated isothiocyanate structure for the
ion at m/z 159 (see Scheme 2). As similar appearance energies
are found for the fragment ion of [2+H]+ at m/z 143 and the
respective fragment ion generated from [1+H]+at m/z 159, both
corresponding to loss of CD3NH2, a nucleophilic attack of the
amide oxygen onto either the urea carbonyl in the former and
the attack on the thiocarbonyl in the latter is unlikely because
the double bond of the thiocarbonyl-moiety in 1 is much less
polarized than the carbonyl moiety in 2. This conclusion also
points toward the open chain protonated isocyanate structure
of the product ion at m/z 143 and the analogous isothiocyanate
structure for the ion at m/z 159.
An important conclusion from these schemes is that the
migration of the ionizing proton along the molecule backbone,
(2) The rate-determining steps are migrations of the ionizing
proton upon CA, which precedes dissociation events of the
protonated molecular ions of compounds 1 and 2. Hence,
the appearance energies taken from the QIT-MS2 breakdown
curves correspond to these transition states and therefore may
not provide any information on the actual fragmentation path-
ways and the respective reaction mechanisms of the precursor
ions examined in the QIT-MS2 experiments.
These assumptions and hypotheses are now probed in more
detail by theory.
3.3. Computational analysis of the reaction pathways
Scheme 6. Computed reaction pathway of the migration of the ionizing proton from
the most basic site in the molecular ion [2+H]+ upon CA to the adjacent urea nitrogen
followed by dissociation to products at m/z 143. Dashed lines indicate bonds being
broken or formed. Only key steps are shown.
To further elucidate the fragmentation pathways and to probe
the assumptions drawn on the basis of the QIT-MS2 data set, the

132
F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
CD3NHCXNH2 (X S or O) and of the m/z 117 product to lose ammo-
nia yielding m/z 100. The formation of the m/z 86 product ion is
still being investigated, but the most likely pathway appears to
be nucleophilic attack of the thiourea nitrogen of protonated 1 at
the carbonyl, displacing CH3NH2 and forming the ␥-butyrolactame
derivative isomer of the m/z 162 product ion, which then easily
loses CD3NCS.
4. Conclusions
Results of the QIT-MS2-experiments document the subtle
competition of the fragmentation pathways of the set of struc-
turally related urea and thiourea-compounds. The extensive
examination of the fragmentation behavior demonstrates that
the substitution of sulfur for oxygen in the urea moiety influ-
ences the fragmentation pattern. Additionally, these results stress
the vital importance of the urea moiety on the fragmenta-
tion pathways and the effective functioning of urea-reagents as
CID-labile XL-compounds. However, it is noted that the model-
compounds with thiourea-moieties examined in this study differ
from the characteristic fragmentation behavior found for the actual
thiourea-XL-compounds. The reason for this deviation is not yet
fully understood and is currently being investigated by theory
and additional tandem-MS experiments with other tailor made
compounds.
The energy-dependent dissociation experiments conducted in
a spherical QIT yield comparable if not equal threshold energies
for the major competing fragmentation reactions under investi-
gation. The characteristic fragmentation reactions of the set of
precursor ions lead to product ions for which different isomeric
structures are considered, i.e., 7-membered heterocyclic ring, a
␥-butyrolactame-derivative, or open chain isothiocyanate and iso-
cyanate ion structures. Theoretical PESs and the extensive set of
experimental results provide a consistent picture of the results,
which allows a reliable identification of individual ion structures.
More advanced energetic experiments remain desirable in order to
allow a more quantitative comparison of the computational results
with experiments.
The computational analysis of the fragmentation pathways
by careful scans of the PES yield characteristic transition states
strongly suggesting that the migration of the ionizing proton along
the molecule backbone, which takes place upon collision activation
prior to fragmentation, is the rate-limiting step for the dominant
fragmentation pathways. The computations clearly show that the
initial transition state of the respective proton moving from the
most basic site of the precursor ion (the thiourea-sulfur in 1 and
the urea carbonyl oxygen in 2) to less basic heteroatom sites of the
molecule is the necessary and decisive step that determines the
extent of the charge-driven fragmentation processes that follow.
This step determines the overall energetics of the major fragmen-
tation processes.
Scheme 7. Computed reaction pathway of the migration of the ionizing proton from
the most basic site in the molecular ion [1+H]+ upon CA to the amide nitrogen fol-
lowed by dissociation to products at m/z 162. Dashed lines indicate bonds being
broken or formed. Only key steps are shown.
which takes place upon collision activation prior to fragmen-
tation, is the rate-limiting step that determines the extent of
the charge driven fragmentation processes that follow for both
reactant ions. Furthermore, theory finds they have compara-
ble energy barriers: 138–145 kJ/mol for 1 and 154–159 kJ/mol
for 2 (see Schemes 5 and 6). The predicted critical energies
for the migration of the ionizing proton differ by about 10%,
which is in good agreement with the outcome of the energy
resolved QIT-MS2 studies of the protonated molecular ions of
1 and 2.
Scheme 7 shows the computed pathway for formation of the
product ion at m/z 162 from protonated 1. The first step is proton
transfer from the sulfur to the nitrogen followed by nucleophilic
attack of the sulfur at the carbonyl carbon, which induces loss of
CH3NH2. For protonated compound 2, the analogous pathway to
Scheme 7 was also explored by using potential energy surface scans.
Here, no stable intermediates could be located anywhere along the
surface, which agrees well with experiment as the loss of CH3NH2
is not found in QIT-MS2 of compound 2 (see Fig. 3). This finding
certainly goes back to the fact that the urea carbonyl oxygen of
compound 2 is substantially less nucleophilic than the respective
sulfur of compound 1. Oxygen is substantially more electroneg-
ative than sulfur, and thus the oxygen binds the ionizing proton
more tightly, thereby effectively restricting the mobility of that
proton. Theoretically, the product ion at m/z 162 found in the MS2
spectrum of [1+H]+ is suggested to be a 7-membered heterocyclic
ion structure, whereas the alternative ␥-butyrolactame derivative
structure is 38–60 kJ/mol higher and the acylium ion structure
(formed by simple CN bond cleavage) lies 79–85 kJ/mol higher in
energy. In this process, the products limit the overall energy for
CH3NH2 elimination, lying 136–146 kJ/mol above the GS reactant,
whereas the TS for proton transfer along this PES lies 12–46 kJ/mol
lower in energy, Scheme 7. This critical energy is nearly identi-
cal with that predicted for CD3NH2 loss, which also agrees well
with experiment as the energy resolved QIT-MS2 studies yield sim-
ilar thresholds for CH3NH2 and CD3NH2 losses, Fig. 5. Given the
similar energetics, it appears that the former process is disfavored
because its proton transfer requires formation of a 9-membered
cyclic TS and cyclization of the products, whereas the latter pro-
cess utilizes a 7-membered cyclic TS and forms the more open
isothiocyanate.
Acknowledgments
This work is generously supported by a doctoral fellowship of
the “Cusanuswerk” to L.F. and by financial support by the DFG to
M.S., F.D. and F.F. which is gratefully acknowledged. M.S., L.F., and
F.F. thank Prof. Klaus Meerholz (Department of Chemistry, Insti-
tute of Physical Chemistry, University of Cologne) for allowing us
to use the Bruker Daltonics Esquire 3000 plus QIT-MS instrument
for the series of energy-resolved MS2 experiments. R.W. and P.B.A.
thank the National Science Foundation, Grant No. CHE-1049580,
for support and the Center for High Performance Computing at
the University of Utah for the generous allocation of computer
time.
Finally, the theoretical approach used here has located a rea-
sonable pathway for decomposition of the parent ions to lose

F. Falvo et al. / International Journal of Mass Spectrometry 330–332 (2012) 124–133
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