Fragmentation of 2-aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/jms.4024

We investigated the gas-phase fragmentation reactions of a series of 2-aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The most intense fragment ions were the acylium ions m/z 105 and [M+H–C₆H_6<

Full text of DOI:10.1002/jms.4024

Fragmentation of 2-aroylbenzofuran derivatives by
electrospray ionization tandem mass spectrometry
Herbert J. Dias, Tatiana M. Vieira, Eduardo J. Crevelin, Paulo M. Donate,
Ricardo Vessecchi and Antônio E. M. Crotti*
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Av. Bandeirantes, 3900, Monte Alegre, CEP 14040-901.
Ribeirão Preto, SP, Brazil.
Correspondence to: Antônio Eduardo Miller Crotti.
E-mail: millercrotti@ffclrp.usp.br
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.4024
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Abstract
We investigated the gas-phase fragmentation reactions of a series of 2-aroylbenzofuran
derivatives by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The most
intense fragment ions were the acylium ions m/z 105 and M+H‒C6H6]+, which originated
directly from the precursor ion as a result of two competitive hydrogen rearrangements.
Eliminations of CO and CO2 from [M+H‒C6H6]+ were also common fragmentation processes
to all the analyzed compounds. In addition, eliminations of the radicals •Br and •Cl were
diagnostic for halogen atoms at aromatic ring A, whereas eliminations of •CH3 and CH2O
were useful to identify the methoxyl group attached to this same ring. We used
thermochemical data, obtained at the B3LYP/6-31+G(d) level of theory, to rationalize the
fragmentation pathways and to elucidate the formation of E, which involved simultaneous
elimination of two CO molecules from B.
Keywords: 2-aroylbenzofuran, fragmentation mechanisms, computational chemistry,
benzofuran compounds
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Introduction
Aroylbenzofurans and their derivatives are important scaffolds to develop drugs.[1]
More specifically, interest in several natural and synthetic 2-aroylbenzofurans has risen due
to their broad spectrum of biological effects, including antifungal,[2, 3] antibacterial,[3-5]
9]
antileishmanial,[6] antioxidant,[3] anti-HIV,[7] anti-tumor,[8,
anti-inflammatory,[10]
antiproliferative, and cytotoxic actions.[11] In addition, some 2-aroylbenzufuran derivatives
can be employed as sensors and semiconductors [12, 13] and as probes for -amyloid plaques in
Alzheimer’s Disease.[14]
Over the last two decades, the advent of electrospray ionization mass spectrometry
(ESI-MS) has enabled analysis of natural and synthetic high-molecular-weight compounds
and of thermolabile and non-volatile molecules.[15] Moreover, tandem mass spectrometry
(MS/MS) has allowed scientists to study, understand, and control some aspects of gas-phase
ion chemistry.[16] Currently, ESI-MS/MS has been used to investigate an increasing number
of classes of synthetic and natural products.[17-21] The resulting data have helped to feed the
literature and to identify the structure of these compounds and their metabolites in complex
mixtures by liquid chromatography tandem mass spectrometry (LC-MS/MS).[15, 16] Despite
efforts to build spectral libraries to identify known compounds on the basis of ESI-MS/MS
data, the lack of systematic studies on the gas-phase fragmentation reactions of some classes
of compounds remains a major barrier.[22, 23]
In spite of the biological activities of 2‒aroylbenzofuran derivatives, data on their
fragmentation are still scarce. To date, Givens and co-workers have reported elimination of
the ring moiety from halogenated benzofuran derivatives ionized by electron ionization (EI)
at 70 eV, followed by CO and HCO• eliminations. Moreover, the benzocyclopropene radical
has been identified as a marker of this class of compounds. Eliminations of •Br and •F in
early stages of fragmentation followed by CO elimination have also been described.[24]
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As part of our ongoing project on the fragmentation reactions of natural products and
25, 26]
synthetic compounds,[17,
and given the scarcity of data about the gas-phase
fragmentation reactions of compounds displaying the benzofuran moiety, here we investigate
the fragmentation pathways of a series of selected 2-aroybenzofuran derivatives by ESI-
MS/MS and multiple-stage mass spectrometry (MSn).
Experimental
Synthesis of 2-aroylbenzofuran derivatives
Compounds 1-5 were obtained by condensation of 2-bromoacetophenone with an o-
hydroxyaldehyde or o-hydroxyketone, according to the methodology described by Yang and
co-workers (Scheme 1).[27] In a two-neck flask (100 mL) connected to a reflux condenser, 2-
bromoacetophenone (1.0 g, 5.0 mmol) and K2CO3 (2.15 g, 15.6 mmol) were dissolved in
DMF (10 mL) for 10 min under inert N2 atmosphere. Next, the o-hydroxyaldehydes II (1.0 g,
6.6 mmol) and IV (1.0 g, 8.1 mmol) or the o-hydroxyketones I (1.0 g, 4.7 mmol), III (1.0 g,
5.0 mmol), and V (1.13 g, 8.3 mmol) were added to 2-bromoacetophenone in DMF, and the
mixture was stirred under reflux (120 °C) for 1 h. The reaction mixture was cooled to room
temperature, and 30 mL of distilled water was added. The mixture was stirred for 5 min and
filtered under reduced pressure. The solids were dried under vacuum, to yield compounds 1
(66.4% yield), 2 (54.7% yield), 3 (70.0% yield), 4 (51.7% yield), and 5 (58.7% yield). All the
1
compounds were identified on the basis of ESI-MS, IR, H, and 13C NMR analyses (see
supplementary information).
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Mass spectrometry analysis
Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses of the
aroylbenzofurans 1-5 were performed on a tandem quadrupole mass spectrometer (Acquity
H-class Xevo TQS, Waters, Milford, MS, USA) equipped with a Z-spray ionization source
operating in the positive ion mode. The samples were dissolved in MeOH/H2O (80:20 v/v)
with 0.1% formic acid; the final concentration of aroylbenzofuran was 1.0 μg mL-1. A trace
of 0.1% formic acid was added, and the sample was infused directly into the ionization
source at a flow rate of 0.1 mL min-1. In the case of the samples used during the deuterium-
labeled experiments, compounds 1-5 were dissolved in MeOH/D2O (1:1 v/v). To maximize
the relative intensity of the peaks corresponding to the protonated aroylbenzofuran
compounds, the capillary and cone potentials were optimized to 3.2 kV and 40 V,
respectively. The ionization source temperature was 150 °C, and the desolvation gas
temperature (N2) was 250 °C. The injected sample volume was 5 µL.
Multiple-stage ESI mass spectrometry (ESI-MSn) analyses were conducted on an Ion
Trap (IT) mass spectrometer (AmaZon Speed, Bruker Daltonics, Bremen, Germany)
operating in the positive ion mode. N2 was used as nebulizing (6 psi) and drying gas (8
mL.min-1, 220 °C). The capillary voltage and the end plate offset were set to 3.5 kV and 500
V, respectively. The analytical mass range was 50-350 m/z.
Computational methods
The most stable conformers of the 2-aroylbenzofuran derivatives were obtained by
conformational analysis and optimization of geometries with the MM2 force field and
molecular dynamics. The most stable conformers were re-optimized on the basis of DFT
calculations, and optimized geometries were obtained for each ion at the B3LYP/6-31+G(d)
level with the Gaussian 03 software.[28] The stationary point was evaluated through
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calculation of vibration frequencies for the same model. To obtain the protonation sites,
proton affinity (PA) was employed as a descriptor to suggest the protonation sites of 2-
aroylbenzofurans 1-5, as observed in some literature reports.[29] The enthalpy of the proton
for the reaction M + H+  MH+ was considered to be 1.48 kcal.mol-1.[30] The fragmentation
mechanisms were proposed on the basis of relative Gibbs energies and enthalpies at 298 K
for all the aroylbenzofuran compounds; the same computational model was used throughout
the experiments. However, the Gibbs energies were used with caution because equilibrium
was not reached during the collision-induced dissociation (CID) experiments. No transition
state was computed because it was assumed that CID supplied the minimal energy for
fragmentation.
Results and Discussion
Structure-fragmentation relationships
In previous studies, our research group showed how important structure-fragmentation
relationships are to elucidate the fragmentation pathways of a series of compounds that share
[31]
the same structural core, such as sesquiterpene lactones
and alkaloids.[17] Ideally, these
relationships are deduced from product ion spectra obtained under an optimum collision
energy (Elab), which is expected to decrease the relative intensity of the protonated molecule
in the MS/MS spectrum by 50% and to increase the relative intensity of the fragment ions
without promoting extensive fragmentation. On the basis of relative intensity (RI, %) versus
Elab (eV) plots, the optimum Elab for aroylbenzofurans 1-5, is 20 eV (Figure 1 for compound
4 and Supplementary material for compounds 1-3 and 5).
Figure 2 shows the product ion spectra of protonated aroylbenzofurans 1-5 at Elab = 20
eV; Table 1 lists the assignments of the main product ions (relative intensity higher than 2%).
Direct elimination of benzene (78 mass units) from the protonated molecule is common to
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compounds 1-5. Product ion B is the most intense in the spectra of aroylbenzofuran
compounds 1-5. Moreover, acylium ion F (m/z 105) and phenyl ion G (m/z 77), produced by
further CO elimination from F, are common fragments to compounds 1-5. Other common
product ions include C, D, and E, which originate from B by elimination of CO (28 mass
units), CO2 (44 mass units), and two CO molecules (56 mass units), respectively. MS3 spectra
support formation of C, D, and E from B (see Supplementary information).
A detailed comparative analysis of the MS/MS spectra of compounds 1-5 allowed us to
identify some diagnostic ions that could help to distinguish the nature of the substituents at
the benzene ring, as depicted in Scheme 2. Eliminations of a halogen (for 1 and 3) and a
methyl radical (for 2) from B produces H (m/z 102) and I (m/z 160), whereas elimination of
formaldehyde (30 mass units) from E is diagnostic for methoxyl group at the benzene ring of
the benzofuran moiety. MS3 experiments also support formation of H and I from B (see
Supplementary information).
Protonation sites and gas-phase reactivity
The relevance of determining the protonation site in gas-phase fragmentation studies
under CID conditions has been extensively discussed. In general, two major possibilities have
been considered: a) the proton is initially attached to the most basic site of the molecule, but
it can migrate to other less basic sites under CID conditions, to trigger fragmentation – this is
known as “mobile proton model”;[32] b) the proton remains attached at the most basic site of
the structure even after CID. In this case, CID only increases the internal energy content.[33]
Also, protonation could occur at different sites, with an excess of the population of species
having the proton attached to the most basic site of the structure. Here, we will use the proton
affinity (PA) values to identify the site that is most susceptible to protonation. We will
explain all the fragmentation routes starting from the most stable precursor ion for each
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compound, which results from proton attachment to the most basic site. We will only
consider the mobile proton model if protonation of less basic sites results in an unstable
species, which could trigger the fragmentation process.
Figure 3 gives the PA values. Besides the oxygen atoms of the furan ring and carbonyl,
carbons C8 and C9 are potential protonation sites, as discussed in previous studies.[34, 35] PA
values indicate that the carbonyl oxygen is the most susceptible to protonation for all the 2-
aroylbenzofuran derivatives. Consequently, we will only consider the species protonated at
the carbonyl oxygen as precursor ion.
Formation of the product ions B-G
Product ions B (A‒78) and F (m/z 105) are the most intense in the product ion spectrum
of 2-aroylbenzofurans 1-5 (Figure 2, Table 1). These acylium ions originate directly from the
precursor ion (i.e., the protonated molecule) by two competitive pathways (I and II, Scheme
2), which result in elimination of benzene (78 u) or C8H3OR1R2R3, respectively. Acylium ion
m/z 105 (F) is widely reported as a marker of compounds displaying carbonyl at benzylic
positions.[36] In the literature, acylium ions originate mostly from charge-induced
‒cleavages, which leads to elimination of a positively-charged leaving group as a neutral
group.[37] However, compounds 1-5 do not contain a leaving group attached to the aromatic
ring of the aroyl moiety that could induce the formation of acylium ion B. Therefore, we
consider that B (A‒78) and F (m/z 105) originate from hydrogen rearrangement from the
carbonyl oxygen to C1’ and C8, respectively (Scheme 3). Data from deuterium-labeled
experiments reveal that all the product ions of [M+D]+ of compounds 1-5 have the same m/z
as the product ions of the protonated molecule (Table 1). Hence, the proton/deuterium
attached to the carbonyl oxygen participates in two competitive hydrogen rearrangements that
generate B and F (m/z 105), as shown in Scheme 3. Competitive formation of acylium ions B
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and F is similar to the competitive formation reported by Hu and co-workers for α, β-
unsaturated aromatic ketones.[38]
We used thermochemical data for fragmentation, estimated by computational chemistry
at the B3LYP/6-31+G(d) level of theory, to investigate differences between the relative
intensities of B (A‒78) and F (m/z 105) in the product ion spectra of protonated 1-5. The G
and H values involved in the formation of B and F are similar; the data demonstrate that
differences between the relative intensities of these ions cannot be associated with their
relative stabilities.
At Elab = 20 eV, the energy transferred to the mass center (Ecom) of protonated 1 (m/z
315), 2 (m/z 253), 3 (m/z 271), 4 (m/z 223), and 5 (m/z 237) by the collision gas (argonium) is
estimated to be 51.9, 62.9, 59.2, 70.1, and 66.5 kcal.mol-1, respectively, as previously
reported for the equation Ecom = Elab [mc/(mc+mi)]).[33, 39] This energy will be enough to
promote dissociation of A into B and F (enthalpy between 30 and 40 kcal.mol-1).
Additionally, the remaining energy content provided by the CID process when Elab is 20 eV
can convert most of fragment ions F (m/z 105) into G (m/z 77). On the other hand, this Elab
can convert only a small fraction of ions B into C (H between 74.4 and 84.1 kcal.mol-1), D
(H between 68.4 and 78.0 kcal.mol-1), and E (H between 49.7 and 58.1 kcal.mol-1). At
least in principle, this could account for the higher relative intensity of B as compared to F in
the product ion spectra of most of the aroylbenzofurans investigated at Elab = 20 eV. In
contrast, product ions B and F have similar relative intensities in the case of aroylbenzofuran
2. The estimated H and G involved in the conversion of [2+H]+ into F is about 4.0‒8.6
kcal.mol-1 higher as compared to the corresponding processes for 1 and 3-5. This difference is
not associated with the relative stability of B, but it results from increased stability of the
protonated molecule of 2 due to the electron-releasing mesomeric effect of the methoxyl
group attached at ring A of compound 2. Thereafter, more energy is necessary to produce F
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from A (H = 40.1 kcal.mol-1 and G = 26.8 kcal.mol-1) for compound 2 than for compounds
1, 3, 4, and 5 (H  31.5‒36.1 kcal.mol-1; G = 18.4‒22.8 kcal.mol-1). Consequently, product
ion F of compound 2 does not dissociate into G (m/z 77), and its relative intensity is therefore
higher as compared to the intensity of F in the product ion spectra of the other compounds.
According to MS3 experiments, the general product ions C, D, and E originate from B,
as illustrated in Scheme 3. These ions result from competitive elimination of CO (28 u), CO2
(44 u), and C2O2 (56 u), respectively. Formation of C involves a single heterolytic cleavage
of the C8‒C10 bond, whereas formation of D from B comprised two steps: (1) formation of
intermediate B’ (H between 36.1 and 62.7 kcal.mol-1), which involves expansion of the
furan ring to produce the δ‒lactone ring; and (2) CO2 elimination, due to contraction of the
δ‒lactone ring to a four-membered ring by a concerted mechanism. CO2 eliminations have
been reported for heterocyclic structures, such as deprotonated flavones, flavonols, and
flavanones.[40, 41] On the other hand, the structure of benzocyclopropenium cation E has been
previously proposed by Givens and co-workers for aroylbenzofuran compounds under
electron ionization mass spectrometry (EI-MS) conditions.[24] Here, we first considered that
product ion E originates from elimination of ethylenedione (C2O2) from B. Elimination of
ethylenedione has also been reported for benzodiazepines investigated by ESI-MS/MS.[42]
However, Schroder and co-workers have described ethylenedione as a short-lived molecule
that is quickly dissociated in two CO unities.[43] On the basis of theoretical calculations, the
enthalpy of E relative to precursor ion A (H = 0.0) is higher than the enthalpy of C and D
relative to A. Nevertheless, the experimental data revealed that product ion E is more intense
in the product ion spectra of 1-5 than C and D. Thus, we compared the H values for the
formation of C, D, and E from B. The H value calculated for the formation of E by
elimination of C2O2 is higher than the H value involved in the formation of C and D by
about 25.7-44.0 kcal.mol-1 and 31.8-50.0 kcal.mol-1, respectively. In contrast, when we
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considered that E originates from elimination of two CO molecules, we found that the
formation of E is energetically favored as compared to the formation of C and D (lower H),
which agrees with its relative intensity in the product ion spectrum of 1-5. Therefore, product
ion E results from elimination of two CO molecules formed by dissociation of C2O2.
Formation of diagnostic ions
Product ion H (m/z 102) emerges only for compounds 1 and 3 and is diagnostic for
halogen at ring A, as depicted in Scheme 4. This ion originates from E by homolytic cleavage
of the C‒X bond (X = Cl or Br). The estimated H and G values for the conversion of E
into H are 88.5 and 78.3 kcal.mol-1 for 1 and 88.5 and 78.8 kcal.mol-1 for 3, respectively.
However, this process violates the even-electron rule.[36, 44, 45] Other similar cases have been
reported for aromatic systems.[46, 47]
Product ions J and I (Scheme 5) are diagnostic for a methoxyl group at aromatic ring A.
Formation of I (m/z 160) involves elimination of a methyl radical by homolytic cleavage of
the C4‒O bond (Scheme 5). In contrast to H, whose formation from E also violates the even-
electron rule,[36, 44, 45] the unpaired electron participates in the resonance with the aromatic
ring, so product ion I is a resonantly stabilized radical cation. Elimination of methyl radical
has also been reported for other classes of compounds displaying methoxy groups attached to
aromatic rings.[45] In addition, elimination of formaldehyde by hydrogen rearrangement yields
fragment ion J (m/z 89), which is also diagnostic for methoxyl group at aromatic ring A.[48]
The H and G involved in the formation of J from B are 75.1 and 39.4 kcal.mol-1,
respectively, whereas the H and G involved in the formation of I from B are 74.8 and 61.8
kcal.mol-1, respectively.
Conclusions
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Formation of acylium ions B and F by two competitive hydrogen rearrangements is the major
fragmentation pathway of the 2-aroylbenzofuran derivatives investigated here. The relative
intensities of B and F in the corresponding CID spectrum are due to the different energy
required for their decomposition rather than their relative stabilities, estimated by
thermochemical data. Moreover, the higher intensity of benzocyclopropene cation E as
compared to vinylic cations C and D can only be understood on the basis of the
decomposition of C2O2 into two CO molecules, as evidenced by Computational Chemistry.
To the best of our knowledge, this is the first study on the gas-phase fragmentation of
protonated benzofuran compounds by ESI-MS/MS and MSn. Studies involving other classes
of benzofuran compounds (e. g., dihydrobenzofuran neolignans) are underway.
Acknowledgements
The authors thank the Brazilian foundations FAPESP (Proc. 2013/20094-0 and 2014/23604-
1) and CNPq (Proc. 141883/2014-6) for fellowships and grants. We would like to thank Prof.
PhD. Carmen Lúcia Cardoso and Juliana Maria de Lima for support with IT-ESI-MSn
analysis (Proc. FAPESP 2014/50299-5).
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Table 1. Product ions of protonated 2-aroylbenzofurans at collision energy of 20 eV. Relative
intensities (%) are in parenthesis. Data from the deuterium-labeled experiments are given in
italics.
Assignment
1
2
3
4
5
A ([M+H]+)
315 (19)
316
253 (23)
254
271 (11)
272
223 (8)
224
237 (8)
238
B (A – C6H6)
237 (100)
237
175 (100)
175
193 (100)
193
145 (100)
145
159 (100)
159
I (B – •CH3)
C (B – CO)
----
160 (1) / 160
147 (4)
147
----
----
----
209 (2)
209
165 (4)
165
117 (2)
117
131 (6)
131
D (B – CO2)
193 (2)
193
131 (2)
131
149 (5)
149
101 (2)
101
115 (4)
115
E (B – 2 CO)
F (A – C8H3OR1R2R3)
H (E – •X)
181 (8)
181
119 (43)
119
137 (13)
137
89 (28)
89
103 (21)
103
105 (19)
105
105 (98)
105
105 (25)
105
105 (62)
105
105 (29)
105
102 (11)
102
----
102 (11)
102
----
----
J (E – CH2O)
G (F – CO)
----
89 (19)
89
----
----
----
77 (6)
77 (30)
77
77 (6)
77 (21)
77 (12)
77
77
77
77
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Scheme 1. Synthesis of 2-aroylbenzofuran compounds 1-5.
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Scheme 2. Structure-fragmentation relationships in protonated aroylbenzofuran compounds
1-5.
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Scheme 3. Formation of the common fragment ions observed in the ESI-MS/MS spectra of
compounds 1-5, their Enthalpies (H), and Gibbs energies (G). The G and H values were
calculated at the B3LYP/6-31 + G(d) level and are in kcal.mol–1.
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Scheme 4. Formation of diagnostic fragment ion H of compounds 1 and 3. Relative
nthalpies (ΔH) and Gibbs energies (ΔG) values are in kcal.mol–1.
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Scheme 5. Formation of diagnostic fragment ions I and J observed in the product ion spectra
of compound 2. Relative enthalpies, bold values, and Gibbs energies, italic values. All the
values are in kcal.mol-1.
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Figure 1. Correlation between relative intensity (%) and collision energy (eV) for compound
4.
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Figure 2. Product ion spectrum of protonated 2-aroylbenzofurans 1-5 (Ar, 20 eV).
Scheme 2. Structure-fragmentation relationships in protonated aroylbenzofuran compounds
1-5.
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Figure 3. Proton affinities (PA) for aroylbenzofurans 1-5. The PA values were calculated at
the B3LYP/6-31 + G(d) level. All the values for PA are in kcal.mol–1.
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