Deprotonated N-(2,4-dinitrophenyl)amino acids undergo cyclization in solution and the gas phase

Article abstract of DOI:10.1016/j.ijms.2011.01.007

The collisionally activated mass spectral fragmentations of N-(2,4-dinitrophenyl)alanine and phenylalanine [M - H]^- may be gas-phase analogs of the base-catalyzed cyclization of N-(2,4-dinitrophenyl) amino acids in aqueous dioxane. This latter reaction is one source of the 2-substituted 5-nitro-1H-benzimidazole-3-oxides, which are antibacterial agents. The fragmentation of both compounds, established by tandem mass spectrometric experiments and supported by molecular modeling using DFT methods, indicate that the [M - H]^- ions dissociate via sequential eliminations of CO ₂ and H₂O to produce deprotonated benzimidazole-N-oxide derivatives. The gas-phase cyclization reactions are analogous to the base-catalyzed cyclization in solution, except that in the latter case, the reactant must be a dianion for the reaction to occur on a reasonable time scale. The cyclization of N-(2-nitrophenyl)phenylalanine, which has one less nitro group, requires a stronger base for the cyclization than the compound with a second nitro group at the 4-position. Following losses of CO₂ and H₂O are expulsions of both neutral molecules and free radicals, the latter being examples of violations of the even-electron ion rule.

Full text of DOI:10.1016/j.ijms.2011.01.007

International Journal of Mass Spectrometry 306 (2011) 232–240
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Deprotonated N-(2,4-dinitrophenyl)amino acids undergo cyclization in solution
and the gas phase
M. Georgea, V. Rameshb, R. Srinivasb, Daryl Giblinc, Michael L. Grossc,∗
a Department of Chemistry, Sacred Heart College, Thevara, Cochin 682013, Kerala, India
b National Center for Mass Spectrometry, IICT, Hyderabad, India
c Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The collisionally activated mass spectral fragmentations of N-(2,4-dinitrophenyl)alanine and phenylala-
nine [M - H]− may be gas-phase analogs of the base-catalyzed cyclization of N-(2,4-dinitrophenyl)amino
acids in aqueous dioxane. This latter reaction is one source of the 2-substituted 5-nitro-1H-
benzimidazole-3-oxides, which are antibacterial agents. The fragmentation of both compounds,
established by tandem mass spectrometric experiments and supported by molecular modeling using DFT
methods, indicate that the [M - H]− ions dissociate via sequential eliminations of CO2 and H2O to produce
deprotonated benzimidazole-N-oxide derivatives. The gas-phase cyclization reactions are analogous to
the base-catalyzed cyclization in solution, except that in the latter case, the reactant must be a dianion
for the reaction to occur on a reasonable time scale. The cyclization of N-(2-nitrophenyl)phenylalanine,
which has one less nitro group, requires a stronger base for the cyclization than the compound with a
second nitro group at the 4-position. Following losses of CO2 and H2O are expulsions of both neutral
molecules and free radicals, the latter being examples of violations of the even-electron ion rule.
© 2011 Elsevier B.V. All rights reserved.
Received 10 September 2010
Received in revised form 6 January 2011
Accepted 10 January 2011
Available online 15 January 2011
Keywords:
Deprotonated N-(2,4-dinitrophenyl)amino
acids
Derivatized amino acids
Tandem mass spectrometry
Mechanisms of fragmentation
Gas-phase cyclization
Density functional theory
Ion structure
H
N
O
1. Introduction
H
N
1. NaOH/
60% dioxane
OH
CH3
Methods for synthesis of 2-substituted 5-nitro-benzimidazole-
N-oxides have received significant attention because these
compounds are potential anti-bacterial agents [1]. The synthe-
sis of substituted benzimidazoles involves the construction of the
imidazole ring via cyclization of appropriate N-substituted dini-
troanilines by using sodium hydroxide in aqueous dioxane [2] or
palladium catalysis [3]. In addition, the cyclization of N-substituted
nitroanilines has been accomplished under thermal and photo-
chemical conditions [4,5]. An efficient method for the synthesis
of 2-methyl-5-nitrobenzimidazole-N-oxide involves the cycliza-
tion of N-(2,4-dinitriphenyl)-alanine by using sodium hydroxide in
dioxane [6,7]. On the basis of kinetics and NMR studies of the reac-
tion mixtures, a cyclization and elimination mechanism involving
an intermediate dianion of the dinitrophenylalanine was proposed
[7].
N+
O-
2. H+/H2O
CH3
NO2
O2N
O2N
A mass spectrometer is a “chemical laboratory” for investigat-
ing unimolecular and ion–molecule reactions [8]. The anion of an
amino acid can be generated in the gas-phase by ESI, and its uni-
molecular reactions can be effectively studied by a combination of
accurate mass and tandem mass spectrometric experiments and
by molecular modeling [9]. The mechanisms of unimolecular reac-
tions taking place in the gas phase, however, are not necessarily
the same as those in solution, and this has motivated comparisons
of reactions that take place in solution with those in the gas phase
by using ESI mass spectrometry and molecular orbital calculations.
Examples of negative-ion ESI applications are investigations of the
Smiles rearrangements [10,11] and the gas-phase denitration of
nitro arenes with carbanions [12]. We reported earlier on the gas-
phase Nazarov cyclization by using both empirical positive-ion ESI
mass spectrometry and theoretical methods; in the gas phase, the
mechanism is strictly unimolecular but not so in solution [13].
We report here a mass spectral study using negative-ion ESI of
2,4-dinitrophenylalanine and phenylalanine to explore whether a
gas-phase cyclization reaction occurs for monoanions analogous
∗
Corresponding author at: Department of Chemistry, Washington University in
St. Louis, One Brookings Drive, Campus Box 1134, St. Louis, MO 63130, USA. Tel.: +1
314 935 4814; fax: +1 314 935 4481.
E-mail address: mgross@wustl.edu (M.L. Gross).
1387-3806/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2011.01.007

M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
233
to that of the cyclization in solution. Because ESI deprotona-
tion produces a monoanion whereas the solution-phase reaction
may require a dianion, the gas-phase cyclization is an imper-
fect analog of the condensed-phase reaction. Nevertheless, the
gas-phase reaction should provide insight on both the gas-phase
and solution reactions. Our approach was to investigate the gas-
phase mechanism by using tandem mass spectrometry, d-labeling,
chemical substitution, and molecular modeling. The N-(2,4-
dinitrophenyl)-alanine (1), N-(2,4-dinitrophenyl)-phenylanine (2),
N-(2-nitrophenyl)-phenylalanine (3) and the N-ethylamide of 1 (6)
were selected for study. The cyclizations of 1 and 2 were carried out
in solution, and the benzimidazole-N-oxides 4 and 5 thus formed
were purified and used as reference compounds to establish the
structures of the gas-phase cyclization products. In the only previ-
ous reports on the mass spectrometry of these compounds, electron
ionization and chemical ionization of 2,4-dinitrophenyl derivatives
of amino acids were used for samples introduced to the ion source
with a particle–beam interface [14].
90 V; the temperature of the capillaries for desolvation was 150 ◦C.
All tuning parameters were optimized to achieve maximum sen-
sitivity at the targeted mass resolving power. The high-resolution
CAD mass spectra (both MS/MS and MS3) were recorded at a mass
resolving power of 30,000 for the product ions; collision voltages
for inducing fragmentation of the precursor ions were in the range
of 7–10 V. To track fragmentation pathways, [M - D]− ions were
generated by ESI from 1:1 D2O/acetonitrile mixture, introduced by
direct infusion and analyzed by CA MS/MS and MS3.
2.3. Theoretical calculations
Theoretical calculations were performed to characterize the
potential-energy surface (PES) associated with fragmentation. The
negative ion of 2,4-dinitrophenyl-alanine and its fragments were
chosen instead of the phenylalanine analog based on size. Con-
former spaces for precursors and intermediates were explored
by Monte-Carlo/MMFF molecular mechanisms/dynamics methods.
From these results, structures of precursors, intermediates, and
scans for associated transition states were explored by using the
PM3 semi-empirical [17,18] algorithm (Spartan for Linux:
We are pleased to contribute this article to an honor issue
for Professor Tino Gaumann, who, during his productive career
at Ecole Polytechnique Fédérale de Lausanne, made important
contributions to the field of gas-phase ion chemistry.
NH
COOH
NH
COOH
NH
COOH
CH3
NO2
NO2
O2N
NO2
O2N
2
3
1
O
H
N
H
N
NH
NH
C2H5
CH3
N+
O-
N+
O-
CH3
NO2
O2N
O2N
O2N
4
6
5
2. Experimental
Wavefunction, Inc.). If necessary, scans were also performed by
DFT: B3LYP/6-31+G(d,p). Minima and transition states were re-
optimized by DFT (Density Function Theory, part of Gaussian
98/03 suites [19,20], Gaussian Inc.) to B3LYP/6-31+G(d,p) and con-
firmed by vibrational frequency analysis. In addition, connections
of transition states to minima were examined by inspection, projec-
tions along normal reaction coordinates, and path calculations as
necessary. Single-point energies were calculated at level B3LYP/6-
311++G(3df,2p)//B3LYP/6-31+G(d,p) and scaled thermal-energy
corrections were applied [21]. DFT was selected for high-level cal-
culations because it requires less computational overhead than ab
initio methods and performs adequately [22]. In addition for com-
parison purposes, single-point energies were calculated at levels
B3LYP/and MP2(fc)/6-311+G(2d,p)//B3LYP/6-31+G(d,p), with the
results being averaged [23] and scaled thermal-energy corrections
applied. All results are reported in kJ/mol as enthalpies of formation
relative to a selected, suitable precursor.
2.1. Synthesis
The N-(2,4-dinitrophenyl)amino acids 1 and 2 were synthe-
sized by reacting the amino acids alanine and phenylalanine
with 2,4-dinitrochlorobenzene, by means of a reported pro-
cedure [15,16]. Compound 3 was synthesized by the reaction
between l-phenylalanine and 1-fluoro-2-nitrobenzene [16]. The
5-nitrobenzimidazole-N-oxides
4 and 5 were synthesized by
refluxing the N-(2,4-dinitrophenyl)amino acids with 10% solution
of NaOH in dioxane/water mixture (6:4) following a known pro-
cedure for the synthesis of 4 [7]. Compound 1 was treated with
thionyl chloride followed by ethylamine to yield the N-ethylamide
(6). The structures of synthetic compounds were confirmed by 1
H
NMR, IR and HRMS data (Supplementary data).
2.2. Mass spectrometry
Calculations on radical products require unrestricted model
chemistries, whereas the restricted format was used for all
closed-shell systems. For fragmentation processes that involve
the production of radical products from non-radical precursors,
we employed multiconfiguration SCF calculations, specifically,
CASSCF(6,8)/6-31G(d,p), which is included in the Gaussian 03 suite
[20]. Transition states and minima were confirmed by vibrational
frequency calculations, and thermal energy corrections were scaled
and applied [21].
Formation of the negative-ion precursors, via deprotonation,
was achieved by using ESI from 1:1 H2O/acetonitrile mixture by
direct infusion. ESI MS and tandem mass spectrometric experi-
ments (CA MS/MS and MS3) were performed by using the Thermo
LCQ Deca Ion-Trap (San Jose, CA) at low mass resolving power
or the Thermo LTQ-Orbitrap mass spectrometer (San Jose, CA) at
high mass resolving power; both were operated in the negative-ion
mode. The needle voltages were 3 kV, and the cone voltages were

234
M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
3. Results and discussion
3.1. N-(2,4-Dinitrophenyl)alanine
Collisional activation of the ESI-produced [M - H]− precursor
ion of N-(2,4-dinitrophenyl)alanine (1) (m/z 254) produces a frag-
ment ion of m/z 192 by elimination of the elements of carbonic
acid, H2CO3 or more likely CO2 + H2O (Fig. 1). The formation of the
fragment ion of m/z 210 by expulsion of CO2 from the [M - H]− ion
suggests that the generation of the fragment ion of m/z 192 is likely
stepwise, and this is supported by the observation that CA of the m/z
210 ion yields the m/z 192 product by means of water loss (data not
shown). Accurate masses of the fragment ions confirm the elemen-
tal compositions (Table 1). Furthermore, the CAD mass spectrum
of the [M - H]− also shows fragment ions of m/z 177, 175, 174 and
162, which were also obtained from the m/z 192 intermediate ion
product as seen in an MS3 experiment. The results show that these
products originate, at least in part, from the ion [H–CO2–H2O]−.
The molecular formulae obtained via accurate mass measurement
indicate that the fragment ions of m/z 177, 175, 174 and 162, which
arise from both m/z 254 and 192 ions, are formed by the expulsions
of •CH3, •OH, H2O and CH2O (formaldehyde, not NO), respectively,
from the ion of m/z 192 (Table 1).
The CAD mass spectrum of the [M - H]− ion (m/z 192) of 2-
methyl-5-nitro-1H-benzimidazole-3-oxide (4) (Fig. 2b), obtained
in solution by cyclization of N-(2,4-dinitrophenyl)alanine by using
NaOH in aqueous dioxane, showed the same fragment ions with a
similar pattern of abundances as that obtained in an MS3 exper-
iment whereby the [M - H–CO2–H2O]− ion was interrogated
(Fig. 2a). The close similarity indicates that the structure of the
intermediate ion of m/z 192 is the deprotonated 2-methyl-5-nitro-
1H-benzimidazole-3-oxide (4). In addition, collisional activation
of the [M - D]− precursor ion of d2-N-(2,4-dinitrophenyl)alanine
obtained by ESI from 1:1 D2O/acetonitrile of 1 causes consecutive
losses of CO2 and HDO indicating the sole remaining D, attached to
N, is lost as part of the HDO (Scheme 1). Hence, we propose that
the formation of the m/z 192 fragment ion involves the gas-phase
cyclization of the [M - H]− ion of N-(2,4-dinitrophenyl)alanine in
a reaction analogous to the base-catalyzed cyclization of the same
substrate in solution. The mechanism of the gas-phase cyclization
likely involves stepwise elimination of CO2 and H2O (Scheme 1).
3.2. N-Ethylamide derivative of 1
Collisional activation of the [M - H]− ion (m/z 281) of the
N-ethylamide derivative of 1 (6) formed by negative-ion ESI also
leads to an abundant fragment ion of m/z 192 in addition to frag-
ment ions of m/z 263 (but not an ion of m/z 210), 177, 175 and 162
(Fig. A in Supplementary data and Table 1). These results suggest
either consecutive losses of H2O and then C2H5NCO or a single-step
loss of 89 u (C2H5NHCOOH). An MS3 experiment of the m/z 192
formed from the intermediate indicates it has the same structure
as that obtained from 1 (data not shown).
3.3. N-(2,4-Dinitrophenyl)phenylalanine
The m/z 192 ions, either from collisional activation of the
[M - H]− of N-(2,4-dinitrophenyl)alanine (1) or the N-ethylamide
derivative of 1 (6) or from deprotonation of 2-methyl-5-nitro-1H-
benzimidazole-3-oxide (4) all yield a product ion of m/z 162, which
arises by elimination of formaldehyde. To investigate further this
elimination, we studied N-(2,4-dinitrophenyl)phenylalanine (2), a
molecule in which a benzyl group replaces the methyl group of 1.
Collisional activation of the ESI-generated [M - H]− of m/z 330 yields
a fragment ion of m/z 268 from the elimination of the elements
of carbonic acid, H2CO3 (62 u, likely CO2 + H2O), a fragmentation

M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
162.0307
235
100
90
80
70
60
50
40
30
20
10
0
254.0413
175.0383
177.0175
168.0174
170
180.0536
192.0409
210.0515
210
150
160
180
190
200
220
230
240
250
m/z
Fig. 1. CAD mass spectrum of ESI-produced [M - H]− ion (m/z 254) of N-(2,4-dinitrophenyl)alanine (1) obtained with an LTQ orbitrap mass spectrometer.
162
a
b
162
100
80
60
40
20
0
100
80
60
40
20
0
177
177
175
174
174
160
170
180
160
170
180
m/z
m/z
Fig. 2. The CAD mass spectra of the m/z 192 ion of the: (a) fragment ion from 1 obtained in an MS3 experiment (b) [M - H]− of 2-methyl-5-nitro-1H-benzimidazole-3-oxide
(4) obtained with LCQ Deca mass spectrometer.
process similar to that occurring for 1. No fragment ion from the
elimination of CO2, [M - H–CO2]−, however, is produced, raising the
possibility that elimination of 62 u may be a one-step process. Other
fragment ions are of m/z 251, 250, 177 and 162 formed by subse-
quent losses of •OH, H2O, benzyl radical, and benzaldehyde from
the ion of m/z 268, as verified by MS3 experiments. The formulae
of the fragment ions, as determined by accurate mass measure-
ments, are consistent with the proposed pathways (Table 1). A
minor fragment ion of m/z 175 from the m/z 268 ion arises from
the elimination of phenoxyl radical. The elimination of formalde-
hyde observed in the case of compound 1 becomes the elimination
of benzaldehyde for compound 2, indicating that both compounds
1 and 2 undergo similar cyclization reactions.
aqueous dioxane under reflux conditions for 2 h. The reaction
product was separated, purified and identified to be 2-benzyl-5-
nitro-1H-benzimidazole-3-oxide (5) by NMR and accurate-mass
measurement by ESI MS. To characterize this product, we collision-
ally activated the [M+H]+ (m/z 270) and observed a fragment ion
of m/z 91 (base peak), which is either benzyl or tropylium, con-
firming the presence of the benzyl group in the product. Collisional
activation of the [M - H]− ion (m/z 268) of 2-the cyclized benzyl-
5-nitro-1H-benzimidazole-3-oxide, however, gave a product-ion
spectrum that is not a good match to that of the m/z 268 frag-
ment from 2, obtained by an MS3 experiment (Fig. 3). CA of the
cyclized product yields an abundant fragment ion of m/z 175, aris-
ing from loss of 93 u, probably a phenoxyl radical, whereas the
m/z 268 ion from 2, studied in an MS3 experiment, yields instead
an abundant ion of m/z 177 by the elimination of a benzyl radical,
Encouraged by the occurrence of a gas-phase cyclization, the
cyclization of 2 in solution was attempted by using NaOH in
- CH2R•
m/z 177
-
CO2
NH
N
m/z 175 (251)
m/z 162
-
N
CH2R
H
NO2
CH2R
- CO2
- HOH
- OH•
O2N
O2N
m/z 192 (268)
R = H (1), (C6H5 (2))
O
- RHCO
m/z 254 (330)
Scheme 1. Proposed gas-phase cyclization and fragmentation of compounds 1 and 2.

236
M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
Table 2
a
162
Calculated relative enthalpies of formation and reaction for schemes (kJ/mol).
100
90
80
70
60
50
40
30
20
10
0
Minima
Label
Transition States
Label
ꢀ2Hf/ꢀHrxn
ꢀꢀH‡
A0
A1
A2r
A2
A2x
A3
−6.8
−16.3
115.2
73.2
17.3
0.0
84.7
81.0
112.6
−54.0
48.7
TS(0–1)
−0.9
253.7
134.7
14.4
211.6
80.5
115.2
109.9
114.4
103.6
70.8
100.2
85.2
37.6
64.6
16.1
TS(3–2r)
TS(2r–2x)
TS(2x–P)
TS(2–P)
TS(2–4r)
TS(4r–4)
TS(3–4s)
TS(4s–4)
TS(4–5)
177
A4
A4r
A4s
A4x
A5
A6
A7
251
250
TS(5–9)
−32.7
TS(4–4x)
TS(4x–6)
TS(6–7)
240
221
220
−2.0
175
206
A8
A9
−2.3
−190.0
−99.1
−332.7
−248.3
−74.7
−79.2
−387.3
−1.9
TS(7–8)
160
180
200
m/z
240
260
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
TS(8–12)
TS(9–10)
TS(10–11)
TS(11–12)
TS(9–13)
TS(13–14)
TS(14–15)
TS(15–18)
TS(18–19)
TS(19–20)
TS(20–21)
TS(21–22)
TS(22–12)
−55.1
−0.4
162
b
−2.5
100
90
80
70
60
50
40
30
20
10
0
−23.7
−66.8
−72.8
−176.8
−206.1
−140.8
−175.7
−76.3
−125.4
−106.6
−204.1
−217.9
−230.6
−173.1
−137.6
251
‡
indicate for transition states the equivalent of the relative enthalpy of formation
for minima designated ꢀ2Hf.
with a dianion mechanism [7] that is facilitated by the increased
acidity of the N–H hydrogen owing to the presence of two nitro
groups. Removal of one nitro group (for 3) decreases the acidity of
the N–H hydrogen and slows the reaction of 3 vs. 2.
175
250
221
220
238
160
180
200
240
260
m/z
3.5. Theoretical results
Fig. 3. CAD mass spectra of m/z 268 ion formed as (a) fragment ion from 2 in an
MS3 experiment and (b) as the [M - H]− ion of 2-benzyl-5-nitro-1H-benzimidazole-
3-oxide (5) taken with an LCQ Deca Ion Trap mass spectrometer).
We carried out theoretical calculations to provide more
support for structures and fragmentation pathways. We sur-
veyed the potential energy surface (PES) for minima and
transition states involved in conversion of the precursor [M -
H]− of N-(2,4-dinitrophenyl)alanine (1) and the related N-(2,4-
dinitrophenyl)-ethylamine for the conversion to the intermediate
and only a minor product of m/z 175. Therefore, the m/z 268 frag-
ment ion formed by the gas-phase cyclization likely arises from an
isomeric structure in addition to deprotonated 2-benzyl-5-nitro-
1H-benzimidazole-3-oxide structure.
ion corresponding to the [M
-
H]− of 2-methyl-5-nitro-1H-
benzimidazole-3-oxide (4) and its subsequent losses of •OH,
•CH3, H2O and H2CO (Section 2, Schemes 2 and 3, Table 2, and
Supplementary data). We note the loss of radicals from closed-shell
precursors violates the even-electron ion rule, but these processes
are energetically tractable because the resultant radical anions are
relatively stable.
3.4. N-(2-Nitrophenyl)phenylalanine
To understand the role of two electron-withdrawing nitro
groups on the cyclization reaction, we investigated the cyclization
of a mono-nitro compound, N-(2-nitrophenyl)phenylalanine (3), in
the gas phase and in solution. Collisional activation of the [M - H]−
ion (m/z 285) of 3 yields an m/z 223 product ion via elimination
of CO2 and H2O. The m/z 223 ion goes on to generate second-
generation fragment ions of m/z 206, 132, and 117 that arise from
the expulsions of •OH, benzyl radical, and benzaldehyde, respec-
tively. These reactions are analogous to the fragmentations of 1
and 2, suggesting strongly the occurrence of a gas-phase cyclization
reaction even when there is a single nitro group at the 4-position.
This compound, however, does not cyclize in solution under the
conditions of refluxing 10% NaOH in 60% aqueous dioxane, as did 1
and 2 that contain two nitro groups. The cyclization, however, does
occur by refluxing with sodium ethoxide in ethanol for 5 h, yield-
ing 2-benzyl-1H-benzimidazole-3-oxide. The nitro group at the
4-position is clearly important for cyclization in solution, in accord
The initial deprotonated N-(2,4-dinitrophenyl)alanine, [M - H]−
(m/z 254) is comprised a pair of equilibrating forms via proton
transfer (A0, A1 in Scheme 2a) with A1 not only being the more
abundant but also primed for the direct loss of CO2, a process
which requires 110 kJ/mol enthalpy with no reverse-activation
barrier (i.e., no transition state). The resulting intermediate A2
undergoes a proton transfer from the amine N to an O of the 2-
nitro group. The resulting product A4r after bond rotation becomes
A4, which can readily undergo ring closure. Deprotonated N-(2,4-
dinitrophenyl)-ethylamine (A3) reacts similarly by proton transfer
from the ethylamine moiety to an O of the 2-nitro group yield-
ing A4s, which becomes A4 after –NO(OH) group rotation. Both
A4r and A4s exist as minima according to the calculations, but
upon adding thermal energy corrections, become shoulders to the
adjoining transition states, Scheme 3.

M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
237
a
CH3
H
O
C
O
-
O2N
N
H
H
H
NoTS
A1
TS(2-4r)
TS(4s-4)
CH3
CH3
NO2
-
m/z 254
O2N
N
O2N
N
- CO2
H
TS(0-1)
H
CH3
A2
m/z 210
A4r
H
NO2
N
O
O
O
C
O
O2N
N
-
-
H
TS(4r-4)
A0
NO2
TS(3-4s)
-
O2N
N
O2N
N
O2N
N
CH3
CH3
CH3
H H
N
N
N
O
OH
A3
A4s
A4
-
m/z 210
-
m/z 210
O
OH
O
O
Scheme 2a. Proposed structures and fragmentation mechanisms: formation of intermediate A4 (m/z 210). Note: A0, A1, [M - H]− of N-(2,4-dinitrophenyl)alanine (1, m/z
254); A3, [M - H]− of N-(2,4-dinitrophenyl)ethylamine (m/z 210).
The intermediate A4 has an activated nitro group, owing to pro-
tonation of one of the oxygens, and that protonated group initiates
a nucleophilic attack effecting ring closure to cyclic A5, which in
turn loses H2O via 1,2-elimination yielding A9 (Scheme 2b). The
composite reaction trajectory (A2, A3) → A4 → A5 → A9 presents
only modest energetic barriers; the maximum transition state is
of ∼120 kJ/mol relative enthalpy (Scheme 3 and Table 2). We note
that A3 does not directly interconvert to A2, but instead both elim-
inate acetaldehyde via transition states of large energetic barriers
(Supplementary data), processes that occur to a modest extent at
best (Fig. 1). This mechanism is in accord with d-labeling results in
that the single deuterium in the [M - H]− of A1 (1) is eliminated
solely as HDO. In addition, collisional activation of the [M - H]−
of N-(2,4-dinitrophenyl)ethylamine yields results consistent with
this scheme (data not shown).
We conclude that loss of CO2 must precede the loss of
H2O en route to the formation of the deprotonated 2-methyl-
5-nitrobenzimidazole-3-N-oxide of m/z 192 (4, A9), in the case
of deprotonated N-(2,4-dinitrophenyl)alanine. We were unable to
find on the PES any competitive route for the loss of carbonic acid,
OC(OH)2, as a single species. There is precedent for carbonic acid
loss in which a cupric ion mediates the formation of the incipi-
ent carbonic acid species, but in the gas phase, there is no such
stabilization [24]. In addition, we searched for stabilization of the
incipient carbonic acid via by incorporation into a ring structure but
were unsuccessful. Consequently, by analogy CO2 likely precedes
the loss of H2O for N-(2,4-dinitrophenyl)phenylalanine, giving an
intermediate, [M - H–CO2]−, that is of insufficient abundance to be
detected.
One possible reaction trajectory from A4 to A12 is
A4 → A4x → A6 → A7 → A8 → A12 (Scheme 2b), wherein H2O
is eliminated from the acyclic form A4x to yield A6 where the neg-
ative charge is resonant between N and the terminal vinyl C. Attack
of the methide C upon the 2-NO oxygen forms a seven-membered
ring species, A7, which then undergoes ring contraction to A8
followed by stepwise extrusion of H2CO to yield A12. This route
would be competitive for fragmentation from A1 or A3 (Scheme 3).
Another route from A4 to A12 involves deprotonated 2-methyl-
5-nitrobenzimidazole-3-N-oxide of m/z 192 (4, A9 – Scheme 2c)
and would be a candidate for fragmentation of A9 to A12. The
intermediate A9 is the most stabilized intermediate on the PES
up to this point in the proposed mechanism (Scheme 3 and
Table 2). Loss of H2CO from A9 must necessarily involve migra-
tions and/or other rearrangements. In this case, •H transfers from
the methyl group to ON forming A10; this is followed by •OH
back migration to the methylene C forming A11 that eliminates
b
-
N
TS(4-5)
CH3
TS(5-9)
O2N
N
A9
- H2O
m/z 192
CH3
CH3
H
N
O2N
N
OH
O
A4
A5
-
m/z 210
O
OH
TS(4-4x)
H
H
-
CH2
-
TS(4x-6)
- H2O
O2N
N
O2N
N
OH
A4x
A6
m/z 192
NO
N
NO
TS(6-7)
-
CH
CH2
N
N
N
TS(7-8)
H TS(8-12)
H
A12
- H2CO
CH2
CH2
m/z 162
-
N
O
O2N
O2N
O2N
N
-
O
O
A7
A8
A8u
Scheme 2b. Proposed structures and fragmentation mechanisms: from intermediates A4 to A9 and alternate route to A12 of m/z 162.

238
M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
c
-
N
N
-
N
N
-
N
TS(10-11)
TS(11-12)
CH2
CH2
H
- H2CO
N
O
H
O2N
O2N
O2N
OH
A11
*A10
A12
- OH•
*NoTS
m/z 162
TS(9-10)
N
N
-
N
-•
N
O
-•
N
CH3
CH2
N
O2N
O2N
O2N
A17
A16
m/z 175
O
A9
m/z 192
TS(9-13)
*TS(14-17)
m/z 177
- CH3•
-
-
O
N
*TS(15-17)
N
TS(13-14)
- CH3•
CH3
TS(14-15)
-
CH3
N
N
N
O2N
*A14
O2N
O
O
A13
N
O2N
CH3
*A15
Scheme 2c. Proposed structures and fragmentations of m/z 192 ion (4, A9). Notes: *Indicates species that were calculated by CASSCF(6,8)/6-31+G(d,p) to assess transition
states with radical character: barrier height for transition state TS(14–17) of 126 kJ/mol relative to A14 and for TS(15–17), 292 kJ/mol relative to A15.
d
O
-
-
NCO
-
N
N
TS(15-18)
TS(18-19)
H
O
H
CH2
N
O2N
N
O2N
O2N
N
CH3
A15
m/z 192
A18
A20
A19
CH2
H
TS(19-20)
-
N
N
O
CH2
O2N
TS(20-21)
-
N
CH
N
N
N
TS(21-22)
TS(22-12)
H
O
H
A12
- H2CO
O
-
m/z 162
N
CH2
O2N
O2N
O2N
N
-
O
CH2
H2C
A21
A22
A22u
Scheme 2d. Proposed structures and fragmentations of m/z 192 (A15).
H2CO (A9 → A10 → A11 → A12). Competitive fragmentation from
A9, however, leads to the formation of A15, the most stable species
that we discovered on the PES; it is formed by an O migration
from N to the adjacent C forming A14 followed by CH3 moiety
back migration to the N forming A15, on the reaction trajectory:
A9 → A13 → A14 → A15 (Scheme 3). Formation of A15 would be
favored over A11 → A12 starting from A9 (Fig. 2), owing to lower
transition state barriers.
Fragmentation Trajectories
200
100
0
TS(4r-4)
TS(3-4s)
TS(4-5)
A4
A4s
A4r
TS(4x-6)
TS(7-8)
TS(6-7)
A2
A3
TS(8-12)
A7
A8
A5
TS(4s-4)
A6 TS(9-13)
TS(10-11)
TS(4-4x)
TS(11-12)
A1
TS(2-4r)
A13
A4x
-100
-200
-300
-400
A14
TS(14-15)
A10
TS(5-9)
TS(9-10)
TS(13-14)
A9
A12
A15
A11
Reaction Coordinate
dNP-EtA (A3) to A4 Alt. Route A4 to A12
dNP-Ala (A1)to A4, A9, A12
A9 to A15
Scheme 3. Calculated reaction trajectories: [M - H]− (A1) to production of the m/z 162 ion (A12).

M. George et al. / International Journal of Mass Spectrometry 306 (2011) 232–240
239
A candidate fragmentation route consistent with the product-
Appendix A. Supplementary data
abundance patterns obtained via low-energy CAD of A9 involves
fragmentation of A15 (Scheme 2d). This route starts with ring
opening of A15 (A18) followed by proton transfer (A19), group
rotations (A20), and ring formation (A21) and contraction to A22.
The intermediate A22 is similar to A8 but with the CH2O moiety
reversed in attachments to the benzimidazole core, a difference
that is >130 kJ/mol more stable according to calculations. In addi-
tion, the largest transition state barriers on the route from A9 to
A12 through A15 are less by 20 kJ/mol the route through A11; thus
this route would predominate (Table 2).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ijms.2011.01.007.
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4. Conclusions
Collisional activation of the [M
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Acknowledgements
M.G. thanks the Principal, S. H. College, Thevara for providing
infrastructure. R.S. thanks Dr. J. S. Yadav, Director, IICT, Hyderabad,
for facilities. Research at WU was supported by the National Cen-
ters for Research Resources of the NIH, Grant P41RR000954 and
by the Washington University Computational Chemistry Facility,
supported by NSF Grant #CHE-0443501.

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