Loss of benzaldehyde in the fragmentation of protonated benzoylamines: Benzoyl cation as a hydride acceptor in the gas phase

Article abstract of DOI:10.1002/jms.3969

In electrospray ionization tandem mass spectrometry of protonated 1-benzoylamines (1-benzoylpiperadine, 1-benzoylmorpholine, and 1-benzoyl-4-methylpiperazine), the dominant fragmentation pathway was amide bond cleavage to form benzoyl cation and neutral amine. Meanwhile, in their fragmentations, an interesting loss of benzaldehyde (106?Da) was observed and identified to derive from hydride transfer reaction between the benzoyl cation and amine. A stepwise mechanism for loss of 106?Da (benzene and CO) could be excluded with the aid of deuterium labeling experiment. Theoretical calculations indicated that hydride transfers from amines (piperadine, morpholine, and 1-methylpiperazine) to benzoyl cation were thermodynamically permitted, and 1-methylpiperazine was the best hydride donor among the 3 amines. The mass spectrometric experimental results were consistent with the computational results. The relative abundance of the iminium cation (relative to the benzoyl cation) in the fragmentation of protonated 1-benzoyl-4-methylpiperazine was higher than that in the fragmentation of the other 2 protonated 1-benzoylamines. By comparing the fragmentations of protonated 1-benzyl-4-methylpiperazine and protonated 1-benzoyl-4-methylpiperazine and the energetics of their hydride transfer reactions, this study revealed that benzoyl cation was a hydride acceptor in the gas phase, but which was weaker than benzyl cation.

Full text of DOI:10.1002/jms.3969

Loss of benzaldehyde in the fragmentation of protonated
benzoylamines: Benzoyl cation as a hydride acceptor in the gas phase
Yunfeng Chaia,d*, Yunlong Shaob, Lu Wangc, Lin Wangb*
a
Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 South Meiling Road,
Hangzhou 310008, P. R. China
b Department of Chemistry and Chemical Engineering, Beijing University of Technology,
Beijing 100124, P. R. China
c College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China
d Department of Chemistry, Zhejiang University, Hangzhou 310027, P. R. China
Running title: Benzoyl cation as a hydride acceptor
Supporting Information
Additional supporting information may be found in the online version of this article at the
publisher’s web site.
*Corresponding author
Correspondence to: Yunfeng Chai, Tea Research Institute, Chinese Academy of Agricultural
Sciences, Hangzhou, P. R. China. E-mail: chaiyunfeng@tricaas.com
Lin Wang, Department of Chemistry and Chemical Engineering, Beijing University of
Technology, Beijing, P. R. China. E-mail: linwang@bjut.edu.cn
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jms.3969
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Abstract
In electrospray ionization tandem mass spectrometry of protonated 1-benzoylamines
(1-benzoylpiperadine, 1-benzoylmorpholine, and 1-benzoyl-4-methylpiperazine), the
dominant fragmentation pathway was amide bond cleavage to form benzoyl cation and
neutral amine. Meanwhile, in their fragmentations, an interesting loss of benzaldehyde (106
Da) was observed and identified to derive from hydride transfer reaction between the benzoyl
cation and amine. A stepwise mechanism for loss of 106 Da (benzene and CO) could be
excluded with the aid of deuterium labeling experiment. Theoretical calculations indicated
that hydride transfers from amines (piperadine, morpholine and 1-methylpiperazine) to
benzoyl cation were thermodynamically permitted and 1-methylpiperazine was the best
hydride donor among the three amines. The mass spectrometric experimental results were
consistent with the computational results. The relative abundance of the iminium cation
(relative
to
the
benzoyl
cation)
in
the
fragmentation
of
protonated
1-benzoyl-4-methylpiperazine was higher than that in the fragmentation of the other two
protonated 1-benzoylamines. By comparing the fragmentations of protonated
1-benzyl-4-methylpiperazine and protonated 1-benzoyl-4-methylpiperazine and the
energetics of their hydride transfer reactions, this study revealed that benzoyl cation was a
hydride acceptor in the gas phase, but which was weaker than benzyl cation.
Keywords: Hydride transfer, Benzoyl cation, Hydride acceptor, Fragmentation mechanism,
Loss of benzaldehyde
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Introduction
Mass spectrometer is not only versatile in analytical chemistry but also serves as a useful tool
in physical organic chemistry. Benefit from the development of electrospray ionization (ESI)
and other soft ionization methods, various interesting ions can be prepared and stored in the
gas phase. Researches on unimolecular or bimolecular reactions of theses ions have attracted
a lot of interest in recent years because of the requirements from not only mass spectrometry
itself but also other disciplines such as biochemistry and organic chemistry. Although the
gas-phase ion chemistry is not equivalent to the chemistry of ions in solution and sometimes
they are quite different, the results obtained from gas phase often can provide valuable even
crucial information for chemical reactions in solution.[1-3] Furthermore, mass spectrometry
can provide convincing experimental evidence to uncover the intrinsic reactivity of a wide
range of species which cannot be probed by other techniques.[4,5]
Hydride transfer is a fundamental reaction in organic and enzymatic reactions.[6-9]
Studying the properties of donor and acceptor in a hydride transfer reaction is necessary but
difficult to tackle in condensed phase interfered by ion pairing, solvation and aggregation
effects. Gas-phase study is an effective access to characterize the intrinsic properties of
hydride transfer reaction. Proton transfer is quite popular in gas-phase reactions, but hydride
transfer is much less common since the occurrence of such reaction needs proper hydride
donor and acceptor. Carbocation is an important class of hydride acceptor, however, in many
cases, it reacts as a proton donor so that the hydride transfer is usually inhibited by the proton
transfer in the gas phase. The typical hydride-abstracting agents are tert-butyl cation and
benzyl cation in the gas phase. The hydride transfer reaction involving these two hydride
acceptors has been extensively studied by mass spectrometric methods.[10-22] Otherwise,
combining mass spectrometry and theoretical calculation, the significant hydride acceptor
properties of benzaldehyde[23], 1,3,5-triazine[24] and phenylisocyanate[25] have be revealed.
The carbonyl group is a hydride acceptor in the reduction of aldehydes and ketones which is
of fundamental importance in chemistry and biochemistry.[26,27] The hydride affinities of
aldehydes and ketones can be determined by experimental method and theoretical
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calculation.[28,29] Acyl cations are reactive species in the gas phase,[30-33] but hydride
abstraction by a bare acyl cation was rarely reported. In solution reaction, an expected
hydride transfer reaction of cycloheptatriene with benzoyl cation was not really observed in
experiment.[34] Only in the gas-phase ion/molecule reactions between benzoyl cation and
alcohols (butan-2-ol or cyclohexanol), the hydride abstraction (neutral benzaldehyde
formation) was observed.[35,36] However, in similar gas-phase ion/molecule reactions
between benzoyl cation and amines, hydride abstraction was not observed.[37-39] The
alkanoyl cation is unstable in the gas phase which usually undergoes loss of CO or
intramolecular cyclization and it is a much better proton donor than hydride acceptor if it
contains α-H.[39-41] The benzoyl cation possesses considerable stability in the gas phase and
it is not a proton donor. Although the hydride abstraction ability of benzoyl cation seems to
be apparent, this issue has not yet been clarified so far. In the present work, the fragmentation
reactions of selected protonated 1-benzoylamines were studied in tandem mass spectrometry.
Loss of benzaldehyde from protonated 1-benzoylamines was identified and proposed to
undergo a hydride transfer process in the fragmentation reaction. To understand the reaction
mechanism, a combination of experimental and theoretical study was carried out herein.
Experimental
Mass Spectrometry
Accurate mass measurements for 1-benzoyl-4-methylpiperazine (compound 3) were
performed on a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA,
USA) equipped with an electrospray ion source. Nitrogen was used as the sheath gas and
auxiliary gas. The spray voltage was set at 3.5 kV. The resolution was set at 100000 and the
capillary temperature was set at 270 °C. The analyte was introduced into the ion source as a
solution in methanol or methanol-d4 at a concentration of approximately 1 μg mL−1. The [M
+ H]+ or [M + D]+ ions of compound 3 were selected as parent ions and their MS/MS spectra
were acquired with helium as the collision gas at a normalized collision energy (NCE) of
25%. The isolation window was 1 m/z. The mass accuracy was calibrated immediately before
measurements according to manufacturer’s instructions in the positive mode. All spectra were
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analyzed and evaluated using Xcalibur 2.0.
The MS/MS experiments for compounds 1-4 were performed using flow inject analyses
(FIA) on a hybrid quadrupole-orbitrap mass spectrometer coupled with a heated electrospray
ion source (ThermoFischer Scientific, Bremen, Germany). The mobile phase was composed
of 50% A and 50% B at a flow rate of 0.2 mL min−1. A was 0.1% formic acid, and B was
methanol. 3 μL of the sample (1 μg mL−1) was injected into the ESI-MS through FIA. The
MS parameters were as follows: spray voltage 3.5 kV in positive mode, sheath gas (N2) flow
40 arb, auxiliary gas (N2) flow 15 arb, heated capillary temperature 375 °C, auxiliary gas
heater temperature 250 °C, S-lens radio frequency (RF) level 60. The MS/MS spectra were
obtained with nitrogen as the collision gas after isolation of the desired precursor ion. The
isolation window was 1 m/z. A 30% normalized collision energy (NCE) was employed for
fragmentation of the [M + H]+ precursor ions. Data processing was performed with Xcalibur
3.0 software.
Theoretical Calculations
All theoretical calculations were carried out using the Gaussian 03 suit of programs [42]. The
structures of species studied were optimized using density functional theory (DFT) method
with B3LYP/6-311++G(2d,p) and B3LYP/6-31++G(d,p) basis sets. Minima (no imaginary
frequencies) or transition states (one imaginary frequency) were determined by frequency
calculations at the same theoretical level. The minima connected by a given transition
structure were confirmed by intrinsic reaction coordinate (IRC) calculations. In calculations,
the temperature was 298.15 K and the pressure was 1.0 atm. The energies discussed here
were the sum of electronic and thermal energies.
General Synthesis
Synthesis of benzoylamines: Benzoyl chloride (2 mmol), amine (2 mmol) and potassium
carbonate (2.5 mmol) were stirred in acetonitrile (2 ml) at room temperature. After the
completion of the reaction, the solvent was evaporated under reduced pressure, water was
added, and the mixture was extracted with ethyl acetate. The combined organic layer was
washed with brine, dried over Na2SO4, and concentrated to afford the product. The structures
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were confirmed by 1H NMR spectroscopy and mass spectrometry.
1-benzoyl piperadine (1). 1HNMR: (500 MHz, CDCl3): δ (ppm) = 7.39 (m, 5H), 3.72 (t, 2H),
3.35 (t, 2H), 1.68 (t, 4H), 1.52 (t, 2H).
1-benzoyl morpholine (2). 1HNMR: (500 MHz, CDCl3): δ (ppm) = 7.42 (m, 5H), 3.46-3.79
(br, 8H).
1
1-benzoyl-4-methylpiperazine (3). HNMR: (500 MHz, CDCl3): δ (ppm) = 7.40 (m, 5H),
3.81 (t, 2H), 3.45 (t, 2H), 2.50 (t, 2H), 2.35 (t, 2H), 2.33 (s, 3H).
Synthesis of 1-benzyl-4-methylpiperazine: 1-Methylpiperazine (2 mmol) and potassium
carbonate (2.5 mmol) were dissolved in 3 ml tetrahydrofuran and stirred at room temperature.
Benzyl bromide (2 mmol) in tetrahydrofuran was added dropwise. After the completion of the
reaction, the solvent was evaporated under reduced pressure, water was added, and the
mixture was extracted with ethyl acetate. The combined organic layer was washed with brine,
dried over Na2SO4, and concentrated to afford the product. The structures were confirmed by
1H NMR spectroscopy and mass spectrometry.
1
1-benzyl-4-methylpiperazine (4). HNMR: (500 MHz, CDCl3): δ (ppm) = 7.19-7.25 (m,
5H), 3.44 (s, 2H), 2.40 (br, 8H), 2.22 (s, 3H).
Results and Discussion
Some 1-benzoylamines (1-3) and 1-benzyl-4-methylpiperazine (4) were selected as model
compounds (Scheme 1). Table 1 summarizes the concerned product ions generated from the
fragmentation of protonated compounds 1-4, and the related MS/MS spectra are given in
Figure 1. In the fragmentation of the [M + H]+ ion of compounds 1-3, amide bond cleavage
leads to the formation of the dominant product ion, benzoyl cation (C7H5O+, m/z 105), which
is usually one of the main reactions in the fragmentation of protonated amide
compounds.[43-45] The corresponding iminium cations (C5H10N+, m/z 84, C4H8NO+, m/z 86
+
and C5H11N2 , m/z 99) can be observed, though their relative abundances are low. The
formation of these iminium cations is interesting and the mechanism is probably hydride
transfer at first glance.
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In view of the relatively highest abundance of the iminium cation in the fragmentation of
protonated 3 among the three 1-benzoylamines, compound 3 was selected as a model
compound to study the formation mechanism of the iminium cation. The high-resolution
MS/MS spectra of protonated 3 (m/z 205) and deuterated 3 (m/z 206) are shown in Figure 2.
The elemental compositions of the product ions in the fragmentation of protonated 3 (m/z 205)
are listed in Table 2, and the relative errors between the measured mass and the theoretical
mass are all less than 4 ppm. In the fragmentation of protonated 3, protonation at the
methylated N leads to the piperazine ring cracking to generate the product ions m/z 148, 162
and 174. Further fragmentations of these three product ions do not produce m/z 99 (the MS3
spectra are given in Figure S1 in the supporting information). There are two possible routes to
produce the iminium cation (m/z 99) as shown in Scheme 2. Isotope labeling method is very
helpful in mass spectrometry to figure out the fragmentation pathway. For the fragmentation
of the [M + D]+ ion of 3, the hydride transfer mechanism gives rise to the iminium cation
containing a deuteron atom (m/z 100). In the other possible route, the sequential losses of
benzene-d1 and CO lead to the formation of the iminium cation containing no deuteron atom
(m/z 99). In the fragmentation of protonated 1 and 2, loss of benzene takes place (m/z 112 and
114), but in the fragmentation of protonated 3, no loss of benzene is observed (Figure 1). In
+
the fragmentation of deuterated 3, the iminium cation (C5H10DN2 , m/z 100.0976) is formed
+
through losing C6H5CHO and no product ion (C5H11N2 , m/z 99.0913) is formed from losses
of C6H5D and CO (Figure 2). Therefore, the sequential losses of benzene and CO to form the
iminium ion can be excluded with the aid of H/D exchange experiment.
The benzoyl cation can be described in terms of the resonance hybrid contributions
(Scheme 3), which had been elucidated using NMR spectroscopic methods and theoretical
calculations.[46-48] In a hydride transfer reaction, the hydride ion may be released to three
positions of the benzoyl cation, including the carbonyl carbon, the ortho positions of the
phenyl ring, and the para position of the phenyl ring. Among these three positions, the
carbonyl carbon should have the highest hydride affinity because it forms an aromatic
product, benzaldehyde. When the hydride is added to the phenyl ring, the products are much
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less stable than the benzaldehyde (Scheme 4). Consequently, in the fragmentation of
protonated 1-benzoylamines (1-3), the neutral counterpart of the iminium cation should be
benzaldehyde.
Secondary aliphatic amines had been shown to be good hydride donors in the gas phase
when benzyl cation was the hydride acceptor in previous studies.[18,22] Removal of a
hydride ion from the methylene group adjacent to the nitrogen of an amine gives rise to a
very stable iminium cation. DFT calculations were performed to evaluate the energy
requirement of hydride transfer reaction between secondary aliphatic amines and benzoyl
cation and the results are listed in Table 3. Piperadine, morpholine and 1-methylpiperazine
are the corresponding hydride donors. Hydride transfer from these N-containing heterocycles
to the benzoyl cation is thermodynamically allowed. 1-Methylpiperazine is the best hydride
donor among them because all its alkyl hydrogens are hydride sources and the methylene
groups adjacent to the N of tertiary amine exhibit the highest reactivity. In the mass
spectrometric experiments, the iminium cation derived from 1-methylpiperazine has the
highest relative abundance (relative to the benzoyl cation) compared with other two iminium
cations. Thus, the experimental and computational results are consistent with each other. The
iminium cation m/z 99 may contains three isomers as shown in Table 3, but p3-2 is the
preferred isomer in terms of energy. The formation of p3-1 and p3-3 in hydride transfer
reaction is even less favored than that of p1. Consequently, p3-2 is probably the main isomer
of m/z 99 and it is used in the following discussions.
Fragmentation of protonated 4 can generate iminium cation m/z 99 and benzyl cation
+
(C7H7 , m/z 91), as shown in Figure 1. The formation of iminium cations in the fragmentation
of N-benzylated cations is a hydride transfer mechanism between the benzyl cation and the
neutral amine, which has been extensively studied in very recent years.[16-23] DFT
calculation indicates that the hydride transfer reaction between benzyl cation and
1-methylpiperazine is highly exothermic (ΔE = −192.1 kJ mol−1, Table 3). The ΔE of hydride
transfer reaction between benzoyl cation and 1-methylpiperazine is −133.3 kJ mol−1 (Table 1).
Theoretical calculation indicates that benzyl cation is a stronger hydride acceptor than
benzoyl cation, which is also consistent with the mass spectrometric experiments. In the
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fragmentation of protonated 1-benzyl-4-methylpiperazine (compound 4), the relative
abundance of the product ion m/z 99 is much higher than that in the fragmentation of
protonated 1-benzoylamines (Table 1).
To further understand the energetics of the fragmentation of protonated 3, a schematic
potential energy diagram of the fragmentation of protonated 3 is given in Figure 3. The
proton affinity (PA) of tertiary amine is usually higher than that of carbonyl compounds. For
example, the PA of (CH3)(C2H5)2N (971.0 kJ mol-1) is higher than that of C6H5CON(CH3)2
(932.7 kJ mol-1).[49] According to computational results, the energy of the N-protonated 3 is
25.6 kJ mol-1 lower than that of the O-protonated 3. For compound 3, the preferred initial
protonation site should be the methylated N. The chair-conformation of compound 3 can
switch to the boat-conformation with low energy barrier (TS-S1). And then the added proton
can migrate from the methylated N to the amide N through a boat-conformation of piperazine
ring. After amide bond cleavage, the benzoyl cation (m/z 105) is generated directly and the
iminium cation (m/z 99) is formed via hydride transfer reaction. The formation of product ion
p3-2 is preferred than that of product ion p3-1 due to the lower energy barrier and product
energy. The energy threshold for the formation of iminium cation p3-2 is lower than that for
the formation of benzoyl cation, but the relative abundance of benzoyl cation is much higher
than that of p3-2 in the CID experiment. Similar phenomenon was also observed in the
fragmentation of protonated N-benzylpiperidine which produces a benzyl cation and an
iminium cation via hydride transfer.[18] A proposed explanation is that a simple bond
cleavage is faster than the hydride transfer reaction. In the transition states (TS-2 and TS-3),
the amide bond is completely broken and a benzoyl cation is formed (see optimized structures
in Figure 3), therefore the hydride transfer reaction here is analogues to a bimolecular
reaction and it reveals the reactivity of a benzoyl cation. Actually, an ion/neutral complex can
be considered to be involved in this hydride transfer reaction. Benzoyl cation transfer via
ion/neutral complex has been reported in the fragmentation of drug molecules containing
both benzoyl and carboxymethyl groups on an aromatic heterocyclic core.[50,51]
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Conclusions
In mass spectrometry, fragmentation of protonated 1-benzoylamines initially produces
benzoyl cation and amine. Hydride transfer between benzoyl cation and amine leads to the
formation of iminium cation by the loss of benzaldehyde. The theoretically predicted hydride
transfer reactivity of benzoyl cation and amines is in good agreement with the observed mass
spectra. Tertiary amines are better hydride donors than secondary amines and benzoyl cation
is a weaker hydride acceptor than benzyl cation. This study provides a new characterization
of the fundamental benzoyl cation in the gas phase. The hydride transfer mechanism can be
used to interpret the loss of benzaldehyde in the fragmentation of ions containing benzoyl
group.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (21405137,
21605002), the Innovative Research Team in Chinese Academy of Agricultural Sciences
(CAAS-ASTIP-2017-TRICAAS-7), and the Central Institute Basic Scientific Research
Expenses Foundation (1610212016005). Support from the National Supercomputing Center
in Shenzhen for advanced computational research is also acknowledged.
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Scheme 1. The structures of compounds studied.
Scheme 2. The proposed fragmentation mechanism of the [M + D]+ ion of
1-benzoyl-4-methylpiperazine (m/z 206)
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Scheme 3. The resonance of benzoyl cation.
Scheme 4. The computed relative energies of isomers of C7H6O+ using DFT method at
B3LYP/6-311++G(2d,p) theoretical level.
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Figure 1. The MS/MS spectra of the [M + H]+ ion of (a) 1-benzoyl piperadine (m/z
190.1223), (b) 1-benzoyl morpholine (m/z 192.1016), (c) 1-benzoyl-4-methylpiperazine (m/z
205.1333), and (d) 1-benzyl-4-methylpiperazine (m/z 191.1539) using a Q-orbitrap mass
spectrometer (NCE 30%).
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Figure 2. The MS/MS spectra of (a) [M + H]+ ion of 1-benzoyl-4-methylpiperazine (m/z
205.1330) and (b) [M + D]+ ion of 1-benzoyl-4-methylpiperazine (m/z 206.1394) using a
LTQ-orbitrap mass spectrometer. The measured m/z value of each product ion is consistent
with the theoretical one.
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Figure 3. Potential energy surface (PES) of the fragmentation of protonated
1-benzoyl-4-methylpiperazine using DFT at the B3LYP level of theory with the 6-31++G(d,p)
basis set. Relative energies are given in kJ mol−1. The optimized structures of TS-2 and TS-3
are shown above the PES.
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Table 1. Product ions generated in the fragmentation of protonated compounds 1-4 (NCE
30%).
Precursor ion
Product ions
Benzyl cation
Benzoyl cation
105 (21.2) b
105 (100)
105 (100)
-
Iminium cation
84 (0.37)
Ratio a
1.8
[1 + H]+
[2 + H]+
[3 + H]+
[4 + H]+
-
-
86 (0.22)
0.2
-
99 (3.86)
3.9
91 (34.81)
99 (17.61)
50.6
a the ratio of the abundance of iminium cation to that of benzoyl cation or benzyl cation
b m/z (relative abundance %)
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Table 2. Accurate masses and elemental compositions of the product ions in the
fragmentation of protonated 1-benzoyl-4-methylpiperazine (m/z 205.1330).
Measured mass
99.0913
Elemental composition
Theoretical mass
99.0917
Error (ppm)
-4.0
+
C5H11N2
105.0331
148.0752
162.0908
174.0909
C7H5O+
105.0335
-3.8
C9H10NO+
C10H12NO+
C11H12NO+
148.0757
-3.4
162.0913
-3.1
174.0913
-2.3
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Table 3. The relative energies of hydride transfer reactions between benzoyl cation/benzyl
cation and selected secondary amines using DFT method at B3LYP/6-311++G(2d,p)
theoretical level.
Amine
Cation
Iminium cation
Neutral species ΔErelative (kJ mol-1)
Benzoyl cation
Benzaldehyde
Benzaldehyde
Benzaldehyde
Benzaldehyde
Benzaldehyde
Toluene
−121.6
−84.2
p1
Benzoyl cation
Benzoyl cation
Benzyl cation
p2
−112.3
−133.3
−100.9
−192.1
p3-1
p3-2
p3-3
p4
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