Fragmentation of Protonated N-(3-Aminophenyl)Benzamide and Its Derivatives in Gas Phase

Article abstract of DOI:10.1007/s13361-016-1342-z

An ion of m/z 110.06036 (ion formula [C₆H₈NO]⁺; error: 0.32 mDa) was observed in the collision induced dissociation tandem mass spectrometry experiments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement product ion purportedly through nitrogen-oxygen (N–O) exchange. The N–O exchange rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-aminophenyl)-O¹⁸-benzamide, where the rearranged ion, [C₆H₈NO¹⁸]⁺ of m/z 112 was available because of the presence of O¹⁸. Theoretical calculations using Density Functional Theory (DFT) at B3LYP/6-31?g(d) level suggest that an ion-neutral complex containing a water molecule and a nitrilium ion was formed via a transition state (TS-1), followed by the water molecule migrating to the anilide ring, eventually leading to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated amide compounds with electron-donating groups at the meta position, such as, –OH, –CH₃, –OCH_3, –NH(CH₃)₂, –NH-Ph, and –NHCOCH₃, all of which show the corresponding rearranged ions in MS/MS spectra. However, the protonated amide compounds containing electron-withdrawing groups, including –Cl, –Br, –CN, –NO₂, and –CF₃, at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13361-016-1342-z

B American Society for Mass Spectrometry, 2016
J. Am. Soc. Mass Spectrom. (2016) 27:917Y926
DOI: 10.1007/s13361-016-1342-z
RESEARCH ARTICLE
Fragmentation of Protonated N-(3-Aminophenyl)Benzamide
and Its Derivatives in Gas Phase
1
2
3
4
5
Chengli Zu, Sukrit Mukhopadhyay, Patrick S. Hanley, Shijing Xia, Bruce M. Bell,
6
7
5
David Grigg, Jeffrey R. Gilbert, John P. O’Brien
1
Analytical Technology Center, The Dow Chemical Company, Midland, MI 48667, USA
Structured Materials, The Dow Chemical Company, Midland, MI 48674, USA
Process Science, The Dow Chemical Company, Midland, MI 48674, USA
Dow Electronic Materials, The Dow Chemical Company, Newark, DE 19713, USA
Analytical Sciences, The Dow Chemical Company, Midland, MI 48667, USA
Organic Polymers and Organometallics, The Dow Chemical Company, Midland, MI 48674, USA
Dow AgroSciences, The Dow Chemical Company, Indianapolis, IN 46268, USA
2
3
4
5
6
7
+
Abstract. An ion of m/z 110.06036 (ion formula [C
6
H
8
NO] ; error: 0.32 mDa) was
observed in the collision induced dissociation tandem mass spectrometry experi-
ments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement prod-
uct ion purportedly through nitrogen-oxygen (N–O) exchange. The N–O exchange
rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-
1
8
18 +
6 8
aminophenyl)-O -benzamide, where the rearranged ion, [C H NO ] of m/z 112
18
was available because of the presence of O . Theoretical calculations using Density
Functional Theory (DFT) at B3LYP/6-31 g(d) level suggest that an ion-neutral com-
plex containing a water molecule and a nitrilium ion was formed via a transition state
(TS-1), followed by the water molecule migrating to the anilide ring, eventually leading
to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated
amide compounds with electron-donating groups at the meta position, such as, –OH, –CH , –OCH –NH(CH ) ,
3
3,
3 2
–
NH-Ph, and –NHCOCH
protonated amide compounds containing electron-withdrawing groups, including –Cl, –Br, –CN, –NO
CF , at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of
3
, all of which show the corresponding rearranged ions in MS/MS spectra. However, the
2
, and –
3
various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be
enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups.
Keywords: Nitrogen–oxygen exchange, Rearrangement, MS/MS, N-(3-aminophenyl)benzamide, DFT
Received: 23 December 2014/Revised: 11 January 2016/Accepted: 13 January 2016/Published Online: 17 March 2016
systems, electronic systems, and software. Traditionally, MS
is considered to be superior in sensitivity, speed, and
specificity in detecting a chemical species; however, it is
usually a challenging task when one intends to differentiate
isomers of compounds.
Tremendous efforts have been made with MS techniques for
the identification of positional aromatic isomers [1–8]. Unlike
radical ions generated with electron impact (EI) ionization [9],
most of the modern atmospheric ionization (API) methods
produce even-electron gaseous ions. In a typical CID MS/MS
experiment, an ionized precursor is mass selected and subse-
quently activated in the collision cell, leading to fragments that
can provide structural information. The applied energy can be
fine-tuned to generate different fragmentation patterns, which
Introduction
ass spectrometry (MS) has been widely used in many
areas, including environmental analysis, food and drug
analysis, synthetic polymers and bio-polymers etc. Further-
more, it has yet to reach its full strength because of the contin-
uous development in ionization methods, analyzers, detection
M
Electronic supplementary material The online version of this article (doi:10.
1007/s13361-016-1342-z) contains supplementary material, which is available
to authorized users.
Correspondence to: Chengli Zu; e-mail: czu@dow.com

918
C. Zu et al.: Fragmentation of Protonated Amides
could be unique among isomers. In some cases, the generation
of one or more fragments may be diagnostic to one of the
positional isomers. Experimental examples of Bortho effect^
derivatives observed through MS/MS experiments. It is expect-
ed that this work will contribute to the understanding of fun-
damentals of unimolecular ion reactions in gas phase.
[2, 6, 10–21] and Bmeta effect^ [2, 7, 22, 23] of positional
aromatic compounds have been recognized with different mass
spectrometry techniques.
The gas phase unimolecular ion chemistry facilitated by Experimental
MS usually involves formation of reactive intermediates,
Reagents and Solvents
leading to final product ions. Ion-neutral complexes (INC)
have been recognized as an important type of reactive
intermediates, and many times formations of INC are crit-
ical steps of fragmentation pathways of ionized species
All reagents were purchased from Sigma-Aldrich (St. Louis,
MO, USA), Matrix Scientific (Columbia, SC, USA), or
Oakwood Chemical (Estill, SC, USA), and HPLC grade sol-
vents were purchased from Fisher Scientific (Fair Lawn, NJ,
USA). Water used in the eluent was obtained from a EMD
Millipore Water purification system (Darmstadt, Germany),
referred as Milli-Q water.
[
24, 25]. Evidence of INC have been frequently found in
many ion fragmentation reactions with CID experiments
26–32].
The current study investigates ionic fragments of protonated
[
N-(3-aminophenyl)benzamide and its derivatives generated in
MS/MS experiments. Ions formed through the N–O exchange
were observed when there was an electron-donating group
located at the anilide ring. Both experimental and theoretical
studies suggested that a water-nitrilium INC was involved in
the rearrangement mechanism. To the best of our knowledge,
this is the first report of the N–O exchange rearrangement of
protonated N-(3-aminophenyl)benzamide and some of its
18
Preparation of Benzoyl-O -chloride
A procedure similar to that described by Wnuk and co-workers
1
8
was used for the preparation of O -labeled benzoyl chloride.
In this work, the hydrolysis of benzonitrile was conducted
under atmospheric pressure instead of a sealed system as de-
scribed by Wnuk et al. [33].
To a 25 mL two-necked round bottom flask was
reaction mixture was cooled to room temperature and the
1
8
added approximately 1.0 g of O -labeled water. The
flask was sealed with a suba-seal septa that was pierced
with polyethylene tubing. The flask was cooled in an ice
bath and hydrogen chloride gas was bubbled through the
water for 10 min.
volatiles were removed by vacuum via rotary evaporation,
1
leaving a light yellow oil. H NMR spectroscopic analysis
revealed a single molecule and the NMR spectrum matched
that previously reported [33].
The concentrated HCl solution was transferred via
pipette to a 50 mL round bottom flask and 1.78 g of
benzonitrile was added to the reaction mixture. A reflux
condenser was attached to the top of the flask and the
reaction mixture was heated to 100 °C for 20 h. The
mixture was cooled to room temperature and cold sodium
bicarbonate solution was added until the pH was 7–8. The
aqueous layer was extracted and washed with dichloro-
methane (3 × 15 mL). The aqueous solution was acidified
with concentrated HCl until the pH < 3 and the product
was extracted with dichloromethane (3 × 20 mL).The
General Procedure of Preparation of Amide
Compounds
All acyl chloride solutions were freshly prepared in acetonitrile
(ACN) at 1 mg/mL. All amine solutions were freshly prepared in
water (H O) at 1 mg/mL. A portion of each acyl chloride solution
2
was mixed with an amine solution with an equal volume and the
mixture was allowed to stand for about 10 min. Each mixture was
diluted with ACN 10-fold for analysis.
Liquid Chromatography/Mass Spectrometry
combined organic cuts were dried with MgSO
4
and the
(
LC/MS)
1
8
volatiles were removed by vacuum. Benzoic acid-O was
Standard samples labeled BN-(3-aminophenyl)benzamide^
from Matrix Scientific, Oakwood Chemical and Aldrich
were first analyzed with a Thermo Q Exactive Orbitrap
mass spectrometer interfaced with a Thermo-Dionex Ulti-
mate 3000 high performance liquid chromatograph system
collected as a white solid (0.379 g, 18%).
1
8
To a 50 mL round bottom flask was added benzoic acid-O
0.200 g; 1.6 mmol). Thionyl chloride (1 mL) was added and a
(
reflux condenser was attached to the flask. The reaction mix-
ture was placed into an 80 °C oil bath and heated for 1 h. The

C. Zu et al.: Fragmentation of Protonated Amides
919
via a high voltage heated electrospray ionization (HESI)
source operating in the positive ion mode. The HPLC
column used for all LC-MS experiments was an Agilent
Zorbax XDB-C18 (3.5 μ) with a dimension of 150 mm ×
potential energy surface (PES). On the other hand, the
same analysis on the transition state geometries indicated
one imaginary frequency. In the latter case, the
GaussView program was used to visualize the vibrational
mode with imaginary frequency in order to ensure that
the atoms moved along the desired reaction coordinate.
The vibrational analysis was used to compute the enthal-
py (H298) at 298 K by augmenting zero point energy to
the electronic energy, obtained from the optimization
procedure. All calculations were performed using G09
suit of programs [37].
2
0
4
.1 mm. Injection volume used for all experiments was
.2 μL and the column temperature was held at a constant
5 °C. Mobile phases for LC-MS experiments consisted
of H O and ACN with 0.1% formic acid using a constant
2
flow rate of 0.6 mL/min. The gradient started at
8
5:15 H O/ACN for 1 min and ramped to 85% ACN
2
within 10 min and held for 5 min. The column was re-
equilibrated at the initial mobile phase composition for
5
min before the next analysis. The eluent was monitored
with a UV diode array detector set at 254 nm with a
bandwidth of 4 nm. The HESI conditions were as follows:
spray voltage (+): 3500 V; capillary temperature: 320 °C;
sheath gas: 60; auxiliary gas: 20; spare gas: 1; probe
heater temperature: 100 °C; S-lens rf level: 50 V. The
Results and Discussion
Which is the Right N-(3-Aminophenyl)Benzamide?
During the LC analysis of a standard compound, N-(3-
aminophenyl)benzamide obtained from different vendors, the
observed retention times were significantly different for the
Bsame labeled^ compounds. These two standards were pur-
chased from Matrix Scientific and Oakwood Chemicals, re-
spectively. LC chromatograms [Figure S1 in Supplementary
Information (SI)] showed that the sample from Matrix Scien-
tific eluted at 2.02 min whereas the sample from Oakwood
Chemicals eluted at 3.75 min.
A close look at the ESI MS/MS spectra (Figure 1) of the
two standards indicated that the Oakwood Chemical stan-
dard was actually 3-amino-N-phenylbenzamide, whereas
the Matrix Scientific standard was the right compound,
N-(3-aminophenyl)benzamide, 1. Ironically, one of two
batches of BN-(3-aminophenyl)benzamide^ ordered from
Aldrich also turned out to be 3-amino-N-phenylbenzamide,
2, not N-(3-aminophenyl)benzamide, 1.
MS conditions were operated in full MS/data dependent
2
(
dd)-MS mode. The MS conditions were: run time:
1
5 min; full MS resolution: 70,000; AGC target: 3e6;
maximum IT: 100 ms; scan range: 100–1500 m/z; dd-
2
MS resolution: 17,500; AGC target: 1e5; maximum IT:
5
0 ms; loop count: 3; MSX count: 1; TopN: 3; isolation
window: 4.0 m/z; data acquisition and analysis were car-
ried out with the Xcalibur 2.0 software package.
All the amide solutions prepared in house were flow
injected with ACN/H O/formic acid (50:50:0.1) into the
2
MS system. Injection volume was 1 μL. The HESI
conditions were set as below: Spray voltage (+): 3500
V; Capillary Temperature: 320 °C; sheath gas: 5; auxil-
iary gas: 1; spare gas: 0; probe heater temperature: 50
°
C; S-lens rf level: 50 V. The MS conditions were
2
operated in full MS/dd-MS mode. The conditions were:
run time: 1 min; full MS resolution: 70,000; AGC target:
An Unusual Fragment Ion in the ESI MS/MS
Spectrum of N-(3-Aminophenyl)Benzamide
3
e6; maximum IT: 100 ms; scan range: 100–500 m/z; dd-
2
MS resolution: 35,000; AGE target: 1e5; maximum IT:
00 ms; loop count: 3; MSX count: 1; TopN: 3; isola-
1
Five distinctive ion peaks are present in the ESI MS/MS
tion window: 4.0 m/z; fixed first mass: 50.0 m/z. For
pseudo MS experiments, the collision energy in source
was set as 35 eV (unless it was stated) to induce disso-
ciation. The mass accuracy from both MS and MS/MS
experiments are within 4 ppm of error.
spectrum of protonated N-(3-aminophenyl)benzamide, 1
3
(
2
(
top spectrum in Figure 1), which are the ions of m/z
+
13.10226 ([C H N O] , protonated 1), m/z 195.09166
13 13 2
+
ion formula, [C H N ] , ionic fragment after loss of
13 11 2
one H O molecule from protonated 1), m/z 135.05538
2
+
(
ion formula, [C H N O] , ionic fragment after loss of a
7 7 2
phenyl group), m/z 110.06042 (ion formula, [C H NO] ),
and m/z 105.03390 (ion formula [C H O] , benzoyl cat-
+
6
8
Computational Methodology
+
7
5
The structures of all the species in ground and transition
states were optimized using Density Functional Theory
ion). All ions except the ion of m/z 110.06042 were
assigned unambiguously. The ion formula of
+
(
DFT) at B3LYP/6-31 g(d) level. This methodology was
[C H NO] indicates that a previously unknown N–O
6
8
used in the past to predict trends in energetics of various
reactions [34, 35]. In case of adducts, the basis set
superposition error was corrected using the counterpoise
method [36]. The vibrational analysis on the ground state
geometries was performed and the lack of imaginary
frequencies was used to ascertain the minima in the
exchange probably occurred during the CID experiment.
In comparison, dissociation of protonated 3-amino-N-
phenylbenzamide, 2 only produced the ion of m/z
+
94.06562 (ion formula, [C H N] ) and the ion of m/z
6
8
+
120.04451 (ion formula, [C H NO] ) (bottom spectrum
7
6
in Figure 1).

920
C. Zu et al.: Fragmentation of Protonated Amides
Figure 1. MS/MS spectra of protonated N-(3-aminophenyl)benzamide, 1, and protonated 3-amino-N-phenylbenzamide, 2. The
mass spectra were collected at high resolution (~35,000 at m/z 200), so that the elemental compositions of the ions were determined
with high mass accuracy
1
8
MS/MS of N-(3-Aminophenyl)-O -Benzamide
considered to primarily reside on the amido oxygen. This
is supported by the calculated relative enthalpies of the
three different protomers (Figure 3). Evidence for amido
oxygen as the protonation site has been observed with
infrared multiple photon dissociation spectroscopy for
GlyGlyGlyH and AlaAlaAlaH in the gas phase by
Wu and McMahon [38].
To investigate if the second hydrogen was from the
amido hydrogen or from ring A, deuterated N-(3-
To further investigate the hypothesis that the N–O exchange
happened in CID, the MS/MS spectrum of N-(3-
aminophenyl)-O -benzamide (1-O ) was acquired (Figure 2).
Unlike the MS/MS spectrum of protonated 1 shown in Figure 1
top), the m/z values of all the ions except that of the ion with
m/z 195.09161 shifted two mass units upward, which was
consistent with an intramolecular N–O rearrangement.
1
8
18
+
+
(
aminophenyl)benzamide (1-d ) was prepared and the MS/
5
MS/MS of Protonated N-(3-Aminophenyl-d5)
Benzamide
MS spectrum of protonated 1-d was collected (Figure 4)
5
under the same conditions as the nondeuterated material.
From the above experiments, it is reasonable to conclude
that the generation of the rearranged fragment ions is
influenced more by the substitution of ring B than sub-
stitution on ring A. The ion formula of the rearranged
ion of m/z 110 indicated that two hydrogen atoms were
transferred along with the amido oxygen to ring B, while
the N–C bond was broken. The first hydrogen should be
picked up from solution during ionization but it was not
clear if the second hydrogen was from the amido hydro-
gen or from ring A. The proton from solution is
Protonated 1-d was observed with m/z 218.13339 [1-d +
5
5
+
H] . The ion of m/z 200.12286 was formed by loss of one
+
molecule of H O from [1-d + H] . The ion of m/z
2
5
135.05524 was formed by loss of benzene-d from [1-d +
5
5
+
H] . When expanding the mass spectrum at m/z 110, two
isobaric ions were displayed: the ion of m/z 110.06065 was
the rearranged ion, and the other ion of m/z 110.06496 was
the benzoyl-d cation. These results ruled out the possibility
5
of hydrogen transfer from ring A and were consistent with
the amido hydrogen transferring to ring B.
18
18
Figure 2. ESI MS/MS spectrum of protonated N-(3-aminophenyl)- O -benzamide, 1-O

C. Zu et al.: Fragmentation of Protonated Amides
921
Figure 3. (I) Molecular structures and nomenclature of protonated ions; (II) optimized geometries of all the ions and the relative
energies of S-2 and S-3 w.r.tS-1
Water-Nitrilium Ion Complex
proton affinity of the amino than the phenol moiety.
However, it is not known if the final product is int-f
or f because the two protomers are experimentally indis-
tinguishable currently. Further investigation of the trans-
formation from int-f to f is beyond the scope of this
work and will be discussed elsewhere.
Computational results indicated that a water-nitrilium ion
complex intermediate b was formed via a transition
state TS-1 (Scheme 1), which directly followed a hydro-
gen transfer between the amido nitrogen and the amido
oxygen. Calculated relative enthalpies of the intermedi-
ate, transition state, and final products are illustrated in
Figure 5. The INC formed this way could further disso-
ciate to give a nitrilium ion (e, m/z 195) by loss of the
The results suggested that the ions formed due to
cleavage of C–C bond (c in Scheme 1) or C–N bond
(
d) were kinetically more favored compared with the
INC route (here, it is assumed that the kinetics of bond
dissociation is comparable to its thermodynamics). On
the other hand, the INC route led to thermodynamically
stable products; formation of e and f were energetically
downhill compared with c and d. Although, enthalpies of
formation of e and f are comparable, the formation of f
may be favored by entropy, which may be significant
due to high effective temperature of these reactions [39].
To further validate if the water molecule is migrating to the
meta position w.r.t the amino group, from which benzonitrile is
electrostatically bound H O molecule. Alternatively, the
2
H O molecule could also migrate to ring B through the
2
transition state TS-2, followed by cleavage of C–N bond.
As a result, an intermediate product ion of m/z 110,
(
int-f, in which the proton is located on the O atom as
shown in Scheme 1) was formed containing the trans-
ferred O atom. The conformation and the energy of
TS-2 are reported in Scheme 1 and Figure 5. Presum-
ably, int-f tautomerizes to ion f via [1, 5]-proton transfer,
(
which is downhill by 30 kcal/mol), thanks to a higher
Figure 4. ESI MS/MS spectrum of protonated deuterated N-(3-aminophenyl)benzamide, 1-d
5

922
C. Zu et al.: Fragmentation of Protonated Amides
(
c)
(e)
(
a)
(b)
(
d)
(f)
Scheme 1. Schematic of different products formed from ion (a)
3
3
cleaved off, a pseudo MS experiment was carried out by
isomer than for the other two isomers. The MS spectrum of
dissociating the ion of m/z 110 generated from protonated 1
by in-source CID. By comparison, the MS/MS spectra of
protonated ortho-aminophenol, protonated meta-aminophenol,
and protonated para-aminophenol were also collected. As
shown in Figure 6, the MS/MS spectra of protonated
aminophenols display the same ion peaks, except that the peak
intensity of the ion of m/z 92 is relatively higher for the ortho
protonated 1 is almost identical to the spectra of protonated
meta- or para- aminophenol, which is also consistent with the
calculated energies of the three ion isomers (see Supplementary
Figure S5 in SI). Assigning an unambiguous structure to the
ion of m/z 110 was insubstantial due to a lack of sufficient
experimental evidence; however, this observation did ascertain
that the ion of m/z 110 is an isomer of protonated aminophenol,
Figure 5. Molecular structures, barrier enthalpy, and enthalpies of formation of different ions shown in Scheme 1; all energies are in
kcal/mol; the conformations of TS-1 and TS-2 are shown in SI

C. Zu et al.: Fragmentation of Protonated Amides
923
110.06062
110.06058
(
a) 100
(b)100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
92.05019
93.03424
65.03954
65.03956
50
100
m/z
50
100
m/z
110.06059
110.06059
(
c) 100
(d) 100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
6
5.03955 93.03420
65.03955 93.03422
50
100
m/z
50
100
m/z
Figure 6. MS/MS spectra of (a) the ion of m/z 110 generated in-source from protonated N-(3-aminophenyl)benzamide, 1; (b)
protonated ortho-aminophenol; (c) protonated meta-aminophenol; and (d) protonated para-aminophenol
and it was not in disagreement with the proposed structure f
based on the theoretical calculation.
Substitution Effect of Acyl Groups
on the Rearrangement
MS/MS experiments were performed for a group of protonated
analogs of 1, with varying acyl groups under the same condi-
tions (Table 2). The acyl groups are categorized as (I) the
benzoyl with electron-withdrawing substituents on the ring;
Rearrangement of Benzamide with Substituents
Other than Amino
MS/MS spectra of protonated benzamide compounds with
varying substituents at the meta position of ring B were
collected under the same conditions. When substituting the
amino group with a benzamidyl group, a rearranged frag-
ment ion of m/z 214.08607 was observed but with a lower
intensity (1.7%) (Compound 3 in Table 1). Substitution
with an acetamidyl group (Compound 4 in Table 1)
yielded a rearranged fragment ion of m/z 152.07058 with
intensity of 1.0%. It is possible that the ion of m/z 110 was
formed from the ion of m/z 152 via loss of acetyl group.
Substitution with a dimethylamino group yielded the
rearranged ion of m/z 138 (Compound 5, 6.7%). This
fragment ion possibly continued on to dissociate to yield
the ion of m/z 110 (3.9%), which was confirmed by the
(
II) the benzoyl with electron-donating substituents on the ring;
and (III) an acetyl group. The rearranged fragment ion of m/z
10 was observed in every case; however, the ion intensity
1
changed with the type of chemical functional group within the
ring. In general, the intensity decreased with electron-donating
substituents (Compounds 14–16), but increased with electron-
withdrawing groups (Compounds 17–23). Moreover, the rear-
rangement also happened with an aliphatic acyl group like
acetyl (Compound 24). The electronic effects of different sub-
stituents on the rearrangement are currently under investigation
and will be published elsewhere.
To the best of our knowledge, a peak unique for the meta-
anilide isomer via N–O exchange has not been reported in CID
mass spectra previously. A few examples of N–O exchange
have been observed for aromatic nitro compounds in EI and
MS/MS spectra of protonated species generated with a CI
source [40]. In those cases, the loss of nitroso (nitrogen and
oxygen exchange occurred before the loss of nitroso) was
observed and the fragmentation may also be enhanced by the
so-called Bortho effect^ [41]. An observation of Bmeta effect^
on fragmentation of carboxyanilides was made by Attygalle
and co-workers in negative CID spectra [2]. It was found that a
3
pseudo MS experiments (see Supplementary Figure S24
in SI). Substitution with a hydrogen atom (i.e, no substit-
uent), yielded a rearranged ion of m/z 95 but with a very
low intensity of 0.3% (Compound 6). Other substitutions
including –OH, –CH and –OCH at the meta position also
3
3
showed rearranged ions in MS/MS spectra of protonated
molecules (Compounds 7–9); however, when it came to
electron-withdrawing substitutions, no N–O exchange frag-
ment ions were available (Compounds 10–13).

924
C. Zu et al.: Fragmentation of Protonated Amides
Table 1. Comparing the Effect of Various Rearranged Ions with Changing Substituents Located on Ring B. The MS/MS spectra are shown in Supplementary
Figures S2–S12 in SI
Compound
Structure
Observed rearranged ion
m/z, ion formula)
Abundance normalized to
total ion current (%)
(
X =
+
1
3
NH
2
110.06042, [C
6
H
8
NO]
21.6
1.7
+
Ph(CO)NH
214.08607, [C13
H
2
12NO ]
+
4
5
CH
3
(CO)NH
110.06034, [C
52.07060, [C
6
H
8
NO]
1.3
1.0
+
1
8
H10NO
2
]
+
NH(CH
)
3 2
110.06000, [C
38.09129, [C
95.04910, [C
111.04442, [C
109.06522, [C
6
H
8
NO]
3.9
6.7
0.3
2.8
0.8
7.0
-
-
-
-
+
1
8
H12NO]
H
+
6
7
8
9
H
OH
6
7
O]
+
6
H
H
H
7
O
O]
2
]
+
CH
OCH
3
7
9
+
3
125.05964, [C
7
9
2
O ]
10
11
12
13
NO
F
Cl
Br
2
NR
NR
NR
NR
NR = no rearrangement ion observed
–
peak for the (R-CO-NH) anion was only observed for the meta
isomers; however, no rearrangement was involved in this frag-
mentation. The same group also discovered an interesting
Bortho effect^ of N-acylanilines with a halogen substitution at
the ortho position in EI spectra, where a peak specific to the
ortho isomers was observed via loss of a halogen radical [10].
interesting N–O exchange rearrangement via an INC and then
a water migration pathway. The rearrangement is inhibited
when electron-withdrawing groups are placed at the meta po-
sitions to the amido bond on ring B. The rearrangement is
enhanced with an electron-withdrawing group, but is dwarfed
with an electron-donating group on ring A. In the case of ring A
being replaced with an aliphatic group (Compound 24) the
rearrangement ion can still be observed but at a lower abun-
dance. Computational results indicate that the INC route leads
to thermodynamically stable products compared with other ion
products (i.e., ions c and d) generated through competitive
fragmentation pathways. The electronic effects of different
Conclusions
The gas-phase chemistry of protonated N-(3-
aminophenyl)benzamide and its derivatives has been investi-
gated experimentally and theoretically, which shows an
Table 2. Effect of Various Acyl Groups on the Rearrangement. The MS/MS Spectra are Shown in Supplementary Figures S13–S23 in SI
+
Compound
6 8
Observed rearranged ion [C H NO]
Abundance normalized to total ion intensities (%)
Structure
Y =
1
Phenyl
o-Hydroxy phenyl
m-Tolyl
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
21.6
0.1
14
15
16
17
18
19
20
21
22
23
24
15.4
13.1
31.3
26.6
37.1
37.7
31.1
33.5
29.3
16.1
p-Tolyl
m-Chlorophenyl
p-Chlorophenyl
m-Cyanophenyl
p-Cyanophenyl
m-Trifluorophenyl
p-Trifluorophenyl
3,5-Dinitrophenyl
Methyl

C. Zu et al.: Fragmentation of Protonated Amides
925
ethoxybenzamide and 2-ethoxybenzylamine as further examples of com-
plex ortho effects. Org. Mass Spectrom. 11, 773–779 (1976)
18. Smith, J.G., Wilson, G.L., Miller, J.M.: Mass spectra of isopropyl benzene
derivatives. Ortho Effect. Org. Mass Spectrom. 10, 5–17 (1975)
substituent on the rearrangement are currently under
investigation.
1
9. Maquestiau, A., Van Haverbeke, Y., Flammang, R., Pardo, M.C., Elguero,
J.: Electron-impact fragmentation of azoles. V. Isomerization of benzimid-
azole before degradation. Org. Mass Spectrom. 9, 1188–1193 (1974)
Acknowledgments
The authors thank Patrick Kastl, Tianlan Zhang, and Matthias
Ober of Dow Chemical Company for their insightful
discussions.
20. Laseter, J.L., Lawler, G.C., Griffin, G.W.: Influence of methyl substitution
on mass spectra of diphenylmethanes. Analytical applications. Anal. Lett.
6, 735–744 (1973)
2
2
1. Tatematsu, A., Yoshizumi, H., Hayashi, E., Nakata, H., Organic mass
spectrometry. Viii. M-16 and M-17 ions from aromatic n-oxides on electron
impact. Tetrahedron Lett. 2985–2989 (1967)
2. Attygalle, A.B., Ruzicka, J., Varughese, D., Bialecki, J.B., Jafri, S.: Low-
energy collision-induced fragmentation of negative ions derived from or-
tho-, meta-, and para-hydroxyphenyl carbaldehydes, ketones, and related
compounds. J. Mass Spectrom. 42, 1207–1217 (2007)
References
1. Jiang, K., Bian, G., Pan, Y., Lai, G.: Recognizing ortho-, meta-, or para-
positional isomers of s-methyl methoxylphenylmethylenehydrazine
2
dithiocarboxylates by ESI-MS : the positional effect of the methoxyl
2
3. Derocque, J.L., Jochem, M.: Studies in mass spectrometry. IV. Fragmen-
tation of aryltriflates. Substituent effects on [aro]+ ion abundances: looking
for a best suited electron energy in the steady state approach. Experimental
evidence for the 'through bond' stabilization of aryl cations by meta electron
donor substituents. Org. Mass Spectrom. 12, 479–487 (1977)
substituent. Int. J. Mass Spectrom. 299, 13–19 (2011)
2
3
. Attygalle, A.B., Nishshanka, U., Weisbecker, C.S.: "Meta elimination," a
diagnostic fragmentation in mass spectrometry. J. Am. Soc. Mass
Spectrom. 22, 1515–1525 (2011)
. Amundson, L.M., Owen, B.C., Gallardo, V.A., Habicht, S.C., Fu, M.,
Shea, R.C., Mossman, A.B., Kenttamaa, H.I.: Differentiation of
regioisomeric aromatic ketocarboxylic acids by positive mode atmospheric
pressure chemical ionization collision-activated dissociation tandem mass
spectrometry in a linear quadrupole ion trap mass spectrometer. J. Am. Soc.
Mass Spectrom. 22, 670–682 (2011)
24. McAdoo, D.J., Morton, T.H.: Gas-phase analogs of cage effects. Acc.
Chem. Res. 26, 295–302 (1993)
25. Grützmacher, H.-F.: Unimolecular reaction mechanisms: the role of reactive
intermediates. Int. J. Mass Spectrom. Ion Processes 118/119, 825–855 (1992)
26. Sun, H., Chai, Y., Xu, X., Pan, Y.: Intramolecular benzyl cation transfer in
the fragmentation of cinchona alkaloid-based quaternary ammonium cat-
ions. Int. J. Mass Spectrom. 335, 16–21 (2013)
27. Kuck, D., Grützmacher, H.-F., Barth, D., Heitkamp, S., Letzel, M.C.L.: The
role of ion/neutral complexes in the fragmentation of n-benzyl-
(alkylpyridinium) ions. Int. J. Mass Spectrom. Ion Processes 306, 159–
166 (2011)
28. Wang, S., Yu, L., Wu, Y., Guo, C., Zhang, N., Jiang, K.: Gas-phase
f r a g m e n t a t i o n o f p r o t o n a t e d n , 2 - d i p h e n y l - n ' - ( p -
toluenesulfonyl)ethanimidamides: tosyl cation transfer versus proton trans-
fer. J. Am. Soc. Mass Spectrom. 26, 1428–1431 (2015)
29. Hu, N., Tu, Y.-P., Jiang, K., Pan, Y.: Intramolecular charge transfer in the
gas phase: fragmentation of protonated sulfonamides in mass spectrometry.
J. Org. Chem. 75, 4244–4250 (2010)
4
. Nishshanka, U., Attygalle, A.B.: Low-energy collision-induced fragmenta-
tion of negative ions derived from diesters of aliphatic dicarboxylic acids
made with hydroxybenzoic acids. J. Mass Spectrom. 43, 1502–1511 (2008)
. Attygalle, A.B., Bialecki, J.B., Nishshanka, U., Weisbecker, C.S., Ruzicka,
J.: Loss of benzene to generate an enolate anion by a site-specific double-
hydrogen transfer during CID fragmentation of o-alkyl ethers of ortho-
hydroxybenzoic acids. J. Mass Spectrom. 43, 1224–1234 (2008)
. Reddy, P.N., Srikanth, R., Venkateswarlu, N., Rao, R.N., Srinivas, R.:
Electrospray ionization tandem mass spectrometric study of three isomeric
substituted aromatic sulfonic acids; differentiation via ortho effects. Rapid
Commun. Mass Spectrom. 19, 72–76 (2005)
5
6
7
8
9
. Moraes, L.A.B., Sabino, A.A., Meurer, E.C., Eberlin, M.N.: Absolute
configuration assignment of ortho, meta, or para isomers by mass spec-
trometry. J. Am. Soc. Mass Spectrom. 16, 431–436 (2005)
. McAnoy, A.M., Dua, S., Blanksby, S.J., Bowie, J.H.: The loss of co from
the ortho, meta and para forms of deprotonated methyl benzoate in the gas
phase. J. Chem. Soc. Perkin Trans. 2, 1665–1673 (2000)
30. Nguyen, M.T., Lahem, D., Flammang, R.: The gas-phase rnx-no+ (x = o, n,
s) cations: nitroso onium cations versus ion-molecule complexes. Chem.
Phys. Lett. 283, 357–362 (1998)
3
1. Kanawati, B., Joniec, S., Winterhalter, R., Moortgat, G.K.: Mass spectro-
metric characterization of small oxocarboxylic acids and gas phase ion
fragmentation mechanisms studied by electrospray triple quadrupole-ms/
ms-tof system and dft theory. Int. J. Mass Spectrom. 266, 97–113 (2007)
2. Sun, H., Chai, Y., Xu, X., Pan, Y.: Intramolecular benzyl cation transfer in
the fragmentation of cinchona alkaloid-based quaternary ammonium cat-
ions. Int. J. Mass Spectrom. 335, 16–21 (2013)
. McLafferty, F.W., Gohlke, R.S.: Mass spectrometric analysis-aromatic
acids and esters. Anal. Chem. 31, 2076–2082 (1959)
3
3
1
0. Jariwala, F.B., Figus, M., Attygalle, A.B.: Ortho effect in electron ioniza-
tion mass spectrometry of n-acylanilines bearing a proximal halo substitu-
ent. J. Am. Soc. Mass Spectrom. 19, 1114–1118 (2008)
3. Wnuk, S., Chowdhury, S.M., Garcia, P.I., Robins, M.J.: Stereodefined
synthesis of o3'-labeled uracil nucleosides. 3'[17o]-Labeled 2'-azido-2'-
deoxyuridine 5'-diphosphate as a probe for the mechanism of inactivation
of ribonucleotide reductases. J. Org. Chem. 67, 1816 (2002)
1
1. Attygalle, A., Ruzicka, J., Varughese, D., Sayed, J.: An unprecedented
ortho effect in mass spectrometric fragmentation of even-electron negative
ions from hydroxyphenyl carbaldehydes and ketones. Tetrahedron Lett. 47,
4601–4603 (2006)
3
4. Hehre, W.J., Ditchfield, R., Pople, J.A.: Self-consistent molecular orbital
methods. Xii. Further extensions of Gaussian-type basis sets for use in
molecular orbital studies of organic molecules. J. Chem. Phys. 56, 2257–
1
2. Mendes, M.A., Rittner, R., Eberlin, M.N., Suwinski, J., Szczepankiewicz,
W.: Ortho effects in the dissociation of ionized n-chlorophenyl- and n-
bromophenyl-2-aminobenzamidines: Intramolecular aromatic substitution
with cyclization to protonated 2-(2-aminophenyl)-1h-benzimidazoles. Eur.
J. Mass Spectrom. 8, 27–33 (2002)
2
261 (1972)
3
5. Hancock, A.N., Schiesser, C.H.: The kinetics of carbonyl radical ring
closures. Chem. Sci. 5, 1967–1973 (2014)
1
3. Danikiewicz, W.: Electron ionization-induced fragmentation of n-alkyl-o-
nitroanilines: observation of new types of ortho-effects. Eur. Mass
Spectrom. 4, 167–179 (1998)
3
6. Boys, S.F., Bernardi, F.: Calculation of small molecular interactions by
differences of separate total energies—some procedures with reduced er-
rors. Mol. Phys. 19 (1970)
1
4. Ramana, D.V., Sundaram, N., George, M.: Ortho effects in organic mole-
cules on electron impact 14 - concerted and stepwise ejections of so2 and n2
from n-arylidene-2-nitrobenzenesulfenamides. Org. Mass Spectrom. 22,
3
7. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A.,
Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A.,
Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino,
J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda,
R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H.,
Vreven, T., Montgomery, J.A., Jr., Peralta, J.E., Ogliaro, F., Bearpark, M.,
Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R.,
Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S.,
Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E.,
Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann,
R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W.,
140–144 (1987)
1
5. Vijfhuizen, P.C., Dijkstra, G.: The role of the ortho position in the loss of
hydrogen cyanide and some other primary fragmentations of aromatic
aldoximes. Adv. Mass Spectrom. 7B, 1266–1270 (1978)
1
6. Schwarz, H.: Some newer aspects of mass spectrometric ortho effects. Top.
Curr. Chem. 73, 231–263 (1978)
17. Schwarz, H., Wolfschuetz, R.: Mass spectral investigation of organic
nitrogen compounds. Xxii. Elimination of ammonia from 2-

926
C. Zu et al.: Fragmentation of Protonated Amides
Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P.,
Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, Ö., Foresman, J.B.,
Ortiz, J.V., Cioslowski, J., Fox, D.J.: Gaussian, Inc, Wallingford (2009)
8. Wu, R., McMahon, T.B.: Infrared multiple photon dissociation spectrosco-
py as structural confirmation for glyglyglyh+ and alaalaalah+ in the gas
phase. Evidence for amide oxygen as the protonation site. J. Am. Soc. Mass
Spectrom. 129, 11312–11313 (2007)
39. Mitchell Wells, J., McLuckey, S.A.: Collision‐induced dissociation (CID)
of peptides and proteins. In: Burlingame, A.L. (ed.) Methods in enzymol-
ogy, vol. 402, pp. 148–185. Academic Press, San Diego (2005)
40. McLafferty, F.W., Turecek, F.: Interpretation of mass spectra, 4th ed.
University Science Books, Mill Valley (1993)
3
41. Gross, J.H.: Mass spectrometry - a textbook, 2nd ed. Springer, Heidelberg
Dordrecht London New York: (2011)