Electronic structure and gas-phase chemistry of protonated α- And β-quinonoid compounds: A mass spectrometry and computational study

Article abstract of DOI:10.1002/rcm.6519

Rationale: The use of quinonoid compounds against tropical diseases and as antitumor agents has prompted the search for new naturally occurring and synthetic derivatives. Among these quinonoid compounds, lapachol and its isomers (α- and β-lapachone) serve as models for the synthesis of new compounds with biological activity, and the use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis as a tool to elucidate and characterize these products has furnished important information about these compounds. Methods: ESI-MS/MS analysis under collision-induced dissociation conditions was used to describe the fragmentation mechanisms for protonated 1,4-naphthoquinone, 1,2-naphthoquinone, α-lapachone, and β-lapachone. The B3LYP/6-31+G(d,p) model was used to obtain proton affinities, gas-phase basicities, and molecular electrostatic potential maps, thus indicating the probable protonation sites. Fragmentation pathways were suggested on the basis of the relative enthalpies of the product ions. Results: The ESI-MS signals of the cationized molecules of ortho quinonoid compounds were more intense than those of the protonated molecule. Formation of the major product ions with m/z 187 from protonated α- and β-lapachone has been attributed to a retro-Diels-Alder (RDA) reaction. Conclusions: MS/MS studies on lapachol isomers (α- and β-lapachone) will facilitate the interpretation of the liquid chromatography (LC)-MS/MS analysis of new metabolites. MS/MS data on the 1,4-naphthoquinone, 1,2-naphthoquinone, α-lapachone and β-lapachone core will help characterize new derivatives from in vitro/in vivo metabolism studies in complex matrices. The product ions revealed the major fragmentation mechanisms and these ions will serve as diagnostic ions to identify each studied compound. Copyright

Full text of DOI:10.1002/rcm.6519

Research Article
Received: 4 December 2012
Revised: 18 January 2013
Accepted: 18 January 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 816–824
Published online Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rcm.6519
Electronic structure and gas-phase chemistry of protonated
a- and b-quinonoid compounds: a mass spectrometry and
computational study
1
2
1
3
Ricardo Vessecchi *, Flávio S. Emery , Norberto P. Lopes and Sérgio E. Galembeck
1
Núcleo de Pesquisa em Produtos Naturais e Sintéticos, Departamento de Física e Química, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brasil
2
Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São
Paulo, Brasil
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil
3
RATIONALE: The use of quinonoid compounds against tropical diseases and as antitumor agents has prompted the
search for new naturally occurring and synthetic derivatives. Among these quinonoid compounds, lapachol and its
isomers (a- and b-lapachone) serve as models for the synthesis of new compounds with biological activity, and the use
of electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis as a tool to elucidate and characterize these
products has furnished important information about these compounds.
METHODS: ESI-MS/MS analysis under collision-induced dissociation conditions was used to describe the fragmenta-
tion mechanisms for protonated 1,4-naphthoquinone, 1,2-naphthoquinone, a-lapachone, and b-lapachone. The B3LYP/
6
-31+G(d,p) model was used to obtain proton affinities, gas-phase basicities, and molecular electrostatic potential maps,
thus indicating the probable protonation sites. Fragmentation pathways were suggested on the basis of the relative
enthalpies of the product ions.
RESULTS: The ESI-MS signals of the cationized molecules of ortho quinonoid compounds were more intense than those
of the protonated molecule. Formation of the major product ions with m/z 187 from protonated a- and b-lapachone has
been attributed to a retro-Diels-Alder (RDA) reaction.
CONCLUSIONS: MS/MS studies on lapachol isomers (a- and b-lapachone) will facilitate the interpretation of the liquid
chromatography (LC)-MS/MS analysis of new metabolites. MS/MS data on the 1,4-naphthoquinone, 1,2-naphthoquinone,
a-lapachone and b-lapachone core will help characterize new derivatives from in vitro/in vivo metabolism studies in
complex matrices. The product ions revealed the major fragmentation mechanisms and these ions will serve as diagnostic ions
to identify each studied compound. Copyright © 2013 John Wiley & Sons, Ltd.
[
1,2]
The use of quinonoid compounds against tropical diseases
The advent of mass spectrometry (MS) with modern ioni-
[3]
[15]
and as antitumor agents has prompted the search for new
zation sources, such as electrospray ionization (ESI)
matrix-assisted laser desorption/ionization (MALDI),
and
has
[
4]
[5,6]
[16]
naturally occuring and synthetic derivatives.
As a result,
[15,16]
several research groups have focused on the synthesis of new
to gain knowledge
about their reactivity and activity as antitumoral, mollusci-
contributed to the analysis of thermolabile molecules.
[5–7]
[17,18]
ortho- or para-quinonoid compounds,
Sequential MS analysis (MS/MS)
is the main tool to
[8]
elucidate and characterize natural products, and new deriva-
[
9]
[10]
[11]
[18–20]
cidal, leishmanicidal,
and anti-inflammatory
agents.
and its isomers
can serve as model for the synthesis
of new compounds with biological activity. Electrochemical
tives from in vitro biotransformation.
In addition,
[12]
Among quinonoid compounds, lapachol
a- and b-lapachone)
previous knowledge of the fragmentation pathways of a
series of compounds exhibiting a conserved structural core
[
13]
(
is useful for structural elucidation based on spectral
[
14]
[17–20]
methods have been used to characterize these compounds,
libraries.
As a result, information about these com-
[14]
[18,20]
furnishing insight into how these new compounds act.
pounds can be used in metabolomic studies.
ESI in combination with MS/MS analysis can be used to
elucidate the gas-phase chemistry of several classes of natural
[
21,22]
compounds.
increased,
The number of studies on quinones has
because these compounds have interesting
[
21–24]
*
Correspondence to: R. Vessecchi, Núcleo de Pesquisa em
Produtos Naturais e Sintéticos (NPPNS), Departamento
de Física e Química, Faculdade de Ciências Farmacêuticas
de Ribeirão Preto, Universidade de São Paulo, Via do Café,
S/N – CEP, 14040-903,Ribeirão Preto/SP, Brasil.
[25]
electrochemical behavior in the ESI source. The protonated
and deprotonated species of 1,4-naphthoquinone can occur
during ESI analysis,
[21,22]
and the radical anion has also been
[25]
detected.
Several papers have described the gas-phase
E-mail: vessecchi@bol.com.br; vessecchi@usp.br
reactivity of quinonoid compounds by combining ESI studies
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 816–824

Study of protonated a- and b-quinonoid compounds
[
21–25]
with computational chemistry.
However, dissociation of
MS/MS data on the a-lapachone or b-lapachone core will
help characterize new derivatives from in vitro/in vivo
metabolism studies.
the protonated 1,4-naphthoquinone (1) and 1,2-naphthoqui-
none (2) has not been investigated (Fig. 1). Comparison of
the properties (reactivity, protonation, and dissociation) of
ortho-naphthoquinone and para-naphthoquinone provides
information about how conjugation affects these systems
[20,27,28]
EXPERIMENTAL
[26]
and more complex quinonoid moieties.
We have previously reported on the versatile reactivity of
lapachol and its congeners (2-hydroxy-1,4-naphthoquinone
Synthesis
[24]
1,4-naphthoquinone (1) and 1,2-naphthoquinone (2) were
purchased from Sigma-Aldrich Brazil Ltda (Sao Paulo,
Brazil). Lapachol was obtained from Tabebuia avellanedae Lor.
ex Griseb, as described by Paternò.
to a- and b-lapachone [(3) and (4)] was achieved following
derivatives) under ESI and CID conditions.
The major
fragmentation channels were assessed and used in further
studies on its derivatives. The MS/MS investigation of
lapachol isomers, such as a- and b-lapachone (Fig. 1, struc-
tures (3) and (4)) should help create a spectral data bank, to
understand the fragmentation and reactivity of new deriva-
tives of these compounds. In addition, the use of mass spectro-
metry for the elucidation of in vitro and in vivo metabolites
from lapachol has been demonstrated, and a- and b-lapachone
were the major secondary metabolites observed in these
[
32]
Lapachol cyclization
[
33]
the methodology of Hooker,
which gave the a-isomer by
reflux of lapachol with hydrochloric acid/acetic acid (1:4) at
ambient temperature. To obtain the b-isomer, lapachol was
dissolved in sulfuric acid (Scheme 1). The structures were
confirmed by spectroscopic analysis.
[
27,28]
studies.
are available for compounds (1)–(4).
structural determination of (1) and (2) has been reported.
To date, only electron ionization (EI-MS) results
[29,30]
Mass spectrometry analysis
The EI-MS
[30]
The stock solutions of (1), (2), (3), and (4) for MS analysis were
prepared from a 0.05 mg.mL solution of each compound in
acetonitrile/water (9:1 v/v).
The high-resolution (HR) ESI-MS analyses were carried out
on a hybrid quadrupole (Q)/time-of-flight (TOF) mass spec-
trometer (UltroTOF-Q, Bruker Daltonics, Billerica, MA,
USA) fitted with an ESI source that was operated in positive
mode. Samples were directly infused into the ionization
+
•
Compound (1) undergoes consecutive CO eliminations.
The major EI fragments ions occur at m/z 130 and 102, and
the main difference between (1) and (2) is the relative
–1
+
•
+
•
intensity of these ions. Recent studies on the metabolism of
(
4) by ESI-MS/MS and high-resolution analysis have helped
[20,27,28]
describe its metabolites in vitro.
However, the authors
did not elucidate the dissociation mechanisms, and there is a
need for more advanced analysis.
In this work, we describe the fragmentation mechanisms
for protonated 1,4-naphthoquinone (1), 1,2-naphthoquinone
–1
source at a flow rate of 10 mL.min . The source block and
desolvation temperature was 150 C. The accurate masses
ꢀ
were obtained by using sodium trifluoroacetate as the mass
standard, after each analysis, and the proposed formulae for
each ion (protonated and cationized molecules) were based
on errors being less than 5 ppm from the calculated mass.
The voltage applied at the capillary emitter was optimized
(2), a-lapachone (3), and b-lapachone (4) under collision-
induced dissociation (CID). The reactivity sites were deter-
[31]
mined through computational quantum chemical models.
The stability of each product ion was described on the
basis of the relative enthalpies and Gibbs energies calcula-
tions that revealed the major fragmentation mechanisms.
(
2.5 kV), in order to achieve a high relative intensity of proto-
+
+
+
nated [M+H] or cationized molecules ([M+Na] or [M+K] ).
For the MS/MS analysis, the precursor ion (protonated
molecule) was selected with an isolation width of 0.5 m/z
(
1)
(
1)
O
2
units and fragmented by CID, using N as the collision gas.
O
Initial CID spectra in low resolution of the precursor ions
(
1) (2)
O(2)
were obtained at Elab of 5, 10, 15, 20, 25, 30, 35, and 40 eV.
(
4) (3)
[34]
Energy-resolved plots
were constructed from the relative
intensity of the precursor and product ions, for better under-
standing of the dissociation processes undergone by the
protonated molecules.
O(2)
(
1)
(2)
N.M. 158
N.M. 158
O(1)
(1)
O
Computational methods
(
3)
O
O(3)
All the molecules had their geometries optimized by the
[35,36]
B3LYP/6-31+G(d,p) model,
using the Gaussian 03 suite
[
37]
of programs.
quinone
Geometry analysis for 1,4-naphtho-
O(2)
(2)O
[25]
[38]
and 2-hydroxy-1,4-naphthoquinone
obtained
(
3)
(4)
N.M. 242
N.M. 242
Figure 1. Structure of naphthoquinones: (1) 1,4-naphthoquinone;
2) 1,2-naphthoquinone; (3) a-lapachone; (4) b-lapachone.
N.M. are the nominal masses in u. (Atom labels for 1,4- and
(
1
,2-naphthoquinone are shown in Supplementary Fig. S1, see
Supporting Information.)
Scheme 1. Preparation of lapachol derivatives.
Rapid Commun. Mass Spectrom. 2013, 27, 816–824
Copyright © 2013 John Wiley & Sons, Ltd.
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R. Vessecchi et al.
by the B3LYP/6-31+G(d,p) model (Supplementary Table S1,
Supporting Information) showed that the bond lengths
early studies, and the results agreed with thermochemical cal-
[21,22,31,40]
culation data.
For (1) and (2), atomic charge analysis
[39]
agreed with those from the X-ray diffraction experiments.
The B3LYP model also furnished the most exact thermoche-
(Supplementary Table S2, Supporting Information) in combi-
nation with HOMO and MEP (Figs. 2 and 3) confirmed that
the oxygens are the most nucleophilic atoms in the molecules.
For (3) and (4), the most nucleophilic sites were the same
oxygen atoms and C(3), as depicted in Fig. 3. As previous
studies with naphthoquinones had shown that protonation
on C(3) was less favorable (lower values of PA and GB) than
[40]
mical parameters for the quinonoid compound derivatives.
Protonation sites were proposed on the basis of the atomic
[41]
charges described by the Merz-Singh-Kollman (MK) scheme,
the molecular electrostatic potential map (MEP) generated from
the electronic density, HOMO analysis, gas-phase basicities
[31]
[46]
(
GBs), and proton affinities (PAs). The MEP is the potential
that on O(1), O(2) and O(3), PAs and GBs were determined
energy of interaction of a proton with the charge distribution
generated by the nuclei and electrons of a molecule. The
GBs and PAs were obtained by variations of Gibbs energies
for oxygen protonation only. It has been shown that PA and
+
+
and enthalpies for a protonation reaction M+H ! MH ,
[
31]
respectively.
The quantum theory of atoms-in-molecules (AIM)
applied, so as to gain insight into the changes in bond
[42]
was
[
40,43]
strength after protonation.
a representation of the structure of a chemical compound,
whose vertices correspond to the atoms of the compound
The molecular graphs
(
[42]
and edges correspond to chemical bonds)
with the aid of the AIM 2000 software.
were plotted
[44]
The fragmentation pathways were suggested by calcula-
tion of the Gibbs energies and enthalpies at 298.15 K, and
the product ions were proposed. The corresponding transi-
tion state (TS) energies were obtained and reported relative
to that of the respective precursor (named as critical energy),
which was characterized by one imaginary vibrational
[45]
frequency. Molekel 4.3
was employed for visualization of
some of the examined properties.
RESULTS
Protonation sites and electronic structure
To purpose the fragmentation mechanism, we searched for
the most favorable site for proton attachment. Quantum
descriptors were used to describe the protonation site in our
Figure 3. HOMO (highest occupied molecular orbital) for the
quinonoid compounds.
Figure 2. Molecular electrostatic potential map (MEP) for
quinonoid compounds (1)–(4) plotted at the surface of
Scheme 2. Proton affinities (in parentheses) and gas-phase
3
electron density equal to 0.05 e/Bohr . Red regions are
basicities (bold) for the protonated molecules. All the values
–1
attractor to positive charge; as instance, a proton.
are in kcal mol .
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Rapid Commun. Mass Spectrom. 2013, 27, 816–824

Study of protonated a- and b-quinonoid compounds
GB values are important descriptors to interpret protonation
in ESI-MS.
O(2) was indicated as the most nucleophilic site. For (4), the
PA and GB values suggest that protonation again takes place
at the O(3) atom, where a possible HB stabilizes the proto-
nated molecule, as depicted in Fig. 4 for (2). Resonance in
the quinonoid system can also contribute to stabilize this
protonation site. The same explanation applies for the higher
value of PA and GB of (4) than of (3).
[
25,46,47]
According to PA and GB analysis the most reactive site for
all the compounds is the quinonoid carbonyl (Scheme 2). The
basicity of (1) is higher than that of (2), suggesting that proto-
nation occurs between the two oxygen atoms, which can be
stabilized through an intramolecular hydrogen bond (HB)
(Fig. 4). AIM analysis and geometry parameters indicated
formation of an intramolecular HB after protonation (Fig. 4).
2
Mass spectrometry studies and fragmentation mechanisms
The r and r r values for a possible intramolecular HB agree
[48]
with those described by Koch and Popelier.
The ESI-MS spectra of (1) and (2) reveal several differences
(Fig. 5). For (1) the base peak in the ESI-MS spectrum is the
The PA and GB values indicate that the protonation site for
+
(3) is the O(2) atom (Scheme 2), which can be explained by the
[(1)+H] ion at m/z 159.0441 (Table 1). For (2), the base peak
+
mesomeric effect from the lone pair of O(3), as was shown in
is the [(2)+Na] ion at m/z 181.0272. The same observations
[46]
studies on 2-hydroxy-1,4-naphthoquinone derivatives.
apply for the spectra of (3) and (4) (Fig. 5). The easy occur-
+
These results agree with those from the MEP analysis, where
rence of [M+Na] ions for (2) and (4) is due to the presence
of vicinal carbonyls at the quinonoid moiety, which stabilize
the sodiated molecules, through electrostatic interaction with
the lone pairs of the oxygen atoms. Thus, the b-quinonoid
system can act as sequestering agents, interacting with
the cation.
The sodiated dimer of (4) at m/z 507.1768 is more intense
than that of (3) at m/z 507.1767 (Fig. 5 and Table 1), and this
can be attributed to the lone pairs of electrons on the vicinal
[24]
carbonyls, as observed in studies with lapachol.
The same
+
behaviour was observed for the [2M+K] ions.
+
+
The relative ESI-MS intensities of the [M+H] and [M+Na]
ions of (1) and (2) are useful for characterizing these isomers.
The same is true when one wishes to distinguish between (3)
and (4) (Fig. 5). However, these analyses must be conducted
under the same experimental conditions; i.e., pH, cone poten-
tial and capillary energy, because signal suppression can
Figure 4. Geometry for the equilibrium structure (left)
+
and molecular graph (right) of [(2)+H] . The occurrence
of BCP (bond critical point) and BP (bond path) between
O(2)-H--O(1), 2.055 Å, indicates the presence of HB. For more
[
42]
[48]
details, see Bader
and Koch and Popelier.
Intens.
1
59.0441
Intens.
x10
+MS, 10.6min #202
6
5
4
3
2
1
000
000
000
000
000
000
0
181.0272
4
+MS, 15.0min #289
(
1)
(2)
1
1
0
0
.25
.00
.75
.50
1
59.0441
187.0410
0.25
0.00
1
97.0029
m/z
m/z
1
00 150 200 250 300 350 400 450 500
50 100 150 200 250 300 350 400 450 500
Intens.
x10
Intens.
x10
5
243.1016
+MS, 7.8min #148
5
281.0572
+MS, 4.7min #90
(
3)
(4)
2
.5
.0
265.0831
1
1
0
0
.5
.0
.5
.0
2
5
07.1768
1.5
.0
265.0831
43.1016
1
2
281.0577
0.5
0.0
5
23.1506
5
07.1768
m/z
m/z
1
00 200 300 400 500 600 700
100 200 300 400 500 600 700 800
Figure 5. ESI-MS spectra for: (1), (2), (3), and (4) in ACN/H O (2:1).
2
Rapid Commun. Mass Spectrom. 2013, 27, 816–824
Copyright © 2013 John Wiley & Sons, Ltd.
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R. Vessecchi et al.
Table 1. HR-ESI-MS data (accurate masses and elemental formulae) obtained from the ESI-MS spectra of the quinonoid
compounds (1)–(4) (Fig. 5). Values in parentheses are the errors in ppm for the exact mass
+
+
+
+
+
compound
[M+H]
[M+Na]
[M+K]
[2M+Na]
[2M+K]
(1)
(2)
(3)
(4)
159.0441
–
–
–
–
–
–
+
C
10
H
7
O
2
(0)
159.0441
181.0272
197.0029
+
+
+
C
10
H
7
O
2
(0)
C
10
H
6
NaO
2
(9.6)
C
10
H
6
KO
2
(15.2)
243.1016
265.0830
281.0568
507.1767
523.1503
+
+
3
+
3
+
+
C H O (0)
C H NaO (1.9)
C H KO (2.5)
C H NaO (2.2)
C H KO (2.8)
1
5
15
3
15 14
15 14
30 28
6
30 28
6
243.1016
265.0831
281.0572
507.1768
523.1506
+
+
3
+
3
+
6
+
6
C H O (0)
C H NaO (1.6)
C H KO (1.09)
C H NaO (2.0)
C H KO (2.8)
1
5
15
3
15 14
15 14
30 28
30 28
[
(1)+H]+
[(2)+H]+
100
100
m/z 159
m/z 131
m/z 117
m/z 107
m/z 105
m/z 103
m/z 77
m/z 159
m/z 131
m/z 103
m/z 77
8
6
4
2
0
0
0
0
0
80
60
40
20
0
m/z 73
0
10
20
30
40
0
10
20
30
40
Elab (eV)
Elab (eV)
[
(3)+H]+
[(4)+H]+
1
00
100
80
60
40
20
0
m/z 243
m/z 243
m/z 225
m/z 201
m/z 187
m/z 159
m/z 131
m/z 103
m/z 225
m/z 201
m/z 187
m/z 159
m/z 131
m/z 105
m/z 103
8
6
4
2
0
0
0
0
0
0
10
20
30
40
0
10
20
30
40
Elab (eV)
Elab (eV)
+
+
+
+
Figure 6. Energy-resolved plots for compounds: [(1)+H] , [(2)+H] , [(3)+H] , and [(4)+H] . All the
MS/MS spectra are available in Supplementary Figs. S2–S5 (see Supporting Information).
[
49]
occur.
Thus, sequential MS is necessary to describe which
spectra and the energy-resolved plots (Figs. 6 and 7). The
dissociation energies were calculated and the Gibbs energies
and relative enthalpies of each proposed ion were obtained.
Formation of the m/z 131 ion occurs endothermically
[25,26]
isomer is being analyzed.
analysis can help elucidate the fragmentation mechanism
for each compound, and the structure of an unknown com-
pound of this class can be suggested.
to the identification of other congeners is that some are isoba-
ric, such as lapachol
detailed study of the fragmentation of the protonated mole-
cules is mandatory and will be discussed below.
The MS/MS spectra of [(1)+H] and [(2)+H] present simi-
lar product ions (Figs. 6 and 7); some of these ions are diag-
nostic and help distinguish between (1) and (2), which is not
possible with EI-MS.
The ESI-MS/MS spectrum of [(1)+H] contains more ions
than that of [(2)+H] . The ions m/z 117, and 105 are specific
to [(1)+H] and can be used to distinguish between 1,4- and
,2-naphthoquinone. Fragmentation mechanisms for proto-
nated (1) and (2) were proposed based on the ESI-MS/MS
In this context, ESI-MS/MS
[25]
+
+
The major obstacle
through ring contraction of [(1)+H] or [(2)+H] (Schemes 3
and 4). The enthalpies relative to m/z 131 were calculated as
[
24]
[46]
–1
and isolapachol.
Therefore, a more
11.47 and 16.74 kcal mol , respectively. Bayer strain explains
+
the difference in entropy between [(1)+H] and the product
ion with m/z 131 (see relative Gibbs energies and enthalpies
+
+
–1
–1
values for the m/z 131 formation, ΔΔS = 38 cal mol K ).
[
34]
The maximum energy transferred by collision (ECM
)
to an
–
1
ion with m/z 159 is approximately 17 kcal mol at an Elab of
5 eV. These values are close to those calculated for the enthal-
pies in this process (Scheme 4).
[29,30]
+
+
Formation of the ions at m/z 103 and 105 will depend on
1,3-hydrogen migration (Schemes 3 and 4), following colli-
sional activation of m/z 159. The critical energy for this migra-
+
1
–
1
tion is 74.6 kcal mol (Scheme 3), which should be supplied
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Study of protonated a- and b-quinonoid compounds
Intens.
2000
Intens.
131.0
103.1
[(2)+H]+
[
(1)+H]+
5
4
3
2
1
00
00
00
00
00
0
105.0
1
500
000
500
0
1
31.0
1
03.0
72.9
1
1
06.9
159.0
7
7.0
63.0
159.0
7
2.9
m/z
m/z
60
80
100
120
140
160
60
80
100
120
140
160
Intens4.
Intens.
5
x10 [(3)+H]+
243.1
x10 [(4)+H]+
2
43.1
5
187.0
0
0
0
.8
.6
.4
4
3
2
1
187.0
225.0
2
25.0
0.2
201.0
159.0
201.0
1
59.0
1
33.0
0
0.0
m/z
m/z
1
00 120 140 160 180 200 220 240
100 120 140 160 180 200 220 240
+
+
+
2
Figure 7. ESI-MS/MS spectra of [(1)+H] and [(2)+H] at Elab = 15 eV (N ) and [(3)+H] and
+
[
2
(4)+H] at Elab = 10 eV (N ) (all the spectra are in low resolution).
+
Scheme 4. Fragmentation mechanisms for [(2)+H] . Values
in parentheses are the relative Gibbs energies at 298 K. Italic
values are the relative enthalpies at 298 K. Values above
arrows are the critical energies of the transition states (TS).
–1
All the values are in kcal mol , obtained using the B3LYP/
6
-31+G(d,p) model.
+
Scheme 3. Fragmentation mechanisms for [(1)+H] . Values
in parentheses are the relative Gibbs energies at 298 K. Italic
values are the relative enthalpies at 298 K. Values above
arrows are the critical energies of the transition states (TS).
The ESI-MS/MS spectrum of protonated 1,2-naphthoquinone
[(2)+H] displays product ions of m/z 159, 131, 103, and 77
Fig. 7). Thus, the absence of the ions at m/z 105 and 107
+
(
–
1
All the values are in kcal mol , obtained using the B3LYP/
-31+G(d,p) model.
is useful to distinguish protonated 1,2-naphthoquinone
6
+
+
[
(2)+H] from protonated 1,4-naphthoquinone [(1)+H] .
+
For [(2)+H] dissociation, formation of the m/z 131 ion
by an Elab of 20 eV, as observed by the increased intensity of the
m/z 103 ion and decreased intensity of m/z 131 ion (Fig. 6). For-
mation of the m/z 103 ion can take place in two distinct ways: (i)
CO elimination by a ring contraction, or (ii) acylium ion forma-
tion with subsequent CO elimination (Scheme 3).
occurs at higher energy than that of the isobaric ion from
+
[(1)+H] (Scheme 4). The higher relative enthalpies of the
+
ions generated from [(2)+H] confirm this observation, in
agreement with the energy-resolved plot of these ions
+
(Fig. 6). Formation of the m/z 131 ion from [(2)+H] also
Rapid Commun. Mass Spectrom. 2013, 27, 816–824
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

R. Vessecchi et al.
+
Scheme 5. Fragmentation mechanisms for [(3)+H] . Values in parenth-
eses are the relative Gibbs energies at 298 K. Lower values are the rela-
–1
tive enthalpies at 298 K. All the values are in kcal mol , obtained
using B3LYP/6-31+G(d,p) model.
takes place at a higher value of Elab. The values for the
other product ions are also in agreement with the energy-
resolved plot.
Using the energy-resolved plots and CID spectra of the simi-
lar ions obtained from protonated (3) and (4) (Figs. 6 and 7), the
fragmentation mechanisms shown in Schemes 5 and 6 are
for the formation of m/z 159 from (3) are lower than from
(4). The same considerations apply to formation of m/z 131
from m/z 159. However, two pathways can be suggested
for the fragmentation of m/z 159: (i) ring contraction and
CO elimination, or (ii) cleavage of the five-membered
ring with CO elimination and phenylium ion formation
(Schemes 5 and 6).
[
20,27]
proposed for the major ions (m/z 187 and 159). Miao et al+.
carried out a low-resolution MS/MS analysis of [(4)+H] and
reported results similar those described here, i.e., the most
intense ions are m/z 187 and 159. These ions are the same as
those observed in the analysis of protonated lapachol, which
make identification of the precursor protonated molecule
confusing. Only the relative intensity of these product ions in
2
Formation of the m/z 225 ion occurs by H O loss from m/z
243. ESI-MS/MS studies on protonated 2-hydroxy-1,
4-naphthoquinone derivatives showed that water elimination
[25]
[46]
occurs only for molecules with substitution at C(3),
the stability of the ring opening for H
due to
2
O elimination. Studies
on lapachol showed that water elimination occurs in the same
proportion for a- and b-lapachone. On the other hand,
protonation takes place on OH for lapachol, in contrast to
the protonation on other 2-hydroxy-1,4-naphthoquinones,
where the proton is attached at the O(2) atom. Therefore,
[25]
the MS/MS spectra of protonated lapachol,
a-lapachone,
and b-lapachone, recorded under the same conditions, can help
distinguish these congeners.
The proposed mechanisms for the formation of the m/z 187
+
+
ion from [(3)+H] and [(4)+H] are very similar to those that
2
the preferred mechanism was H O elimination from proto-
nated a- or b-lapachone as depicted for 2-hydroxy-1,
[50]
occur for the C ring of isoflavones and flavonoids.
An
[
51]
RDA reaction occurs with elimination of an alkene and for-
mation of a carbonyl system (Schemes 5 and 6, m/z 243 ! 187).
Early ESI-MS/MS and computational chemistry studies of
protonated lapachol and isolapachol showed that this reaction
is the major fragmentation process.
of this step are 34.76 and 50.54 kcal mol for [(3)+H] and
4-naphthoquinone derivatives, using the Grob mechanism,
[46]
which involves ring opening through acylium formation.
[24,46]
The Gibbs energies
–1
+
+
[
(4)+H] , respectively. Analysis of the energy-resolved plot
(
Fig. 6) and the MS/MS spectra (Fig. 7) shows that formation
+
of the m/z 187 ion occurs at a lower energy for [(3)+H] than
for [(4)+H] . An E of 10 eV for [(3)+H] furnishes the m/z
1
+
+
lab
+
87 ion at 100%. For [(4)+H] , this intensity is achieved at an
Elab of 15 eV (Figs. 6 and 7). These results agree with those
described for the Gibbs energies and enthalpies calculated at
the B3LYP/6-31+G(d,p) level.
Thus, the intensity of the m/z 187 ion at a known Elab helps
differentiate (3) and (4). Another difference is the intensity of
other product ions, such as m/z 201 and 159. Formation of the
m/z 159 ion was attributed to dissociation of the m/z 187 ions
through CO elimination (m/z 187 ! 159, Schemes 5 and 6).
Formation of m/z 159 occurs at lower energy for (3) than for
+
Scheme 6. Fragmentation mechanisms for [(4)+H] . Values
in parentheses are the relative Gibbs energies at 298 K. Lower
values are the relative enthalpies at 298 K. All the values are
(
4). Analysis of the energy plots and the calculated values
for Gibbs energies and relative enthalpies corroborates this
attribution. Therefore, the relative enthalpy values (ΔH298
–1
)
in kcal mol , obtained using B3LYP/6-31+G(d,p) model.
wileyonlinelibrary.com/journal/rcm
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 816–824

Study of protonated a- and b-quinonoid compounds
CONCLUSIONS
F. C. De Abreu, A. V. Pinto, R. C. Montenegro, L. V. Costa-Lotufo,
M. O. F. Growth inhibitory effects of 3 ’-nitro-3-phenylamino nor-
beta-lapachone against HL-60: a redox-dependent mechan-
ism. Toxicol. Vitro 2012, 26, 585.
ESI-MS studies on 1,2-naphthoquinone and 1,4-naphthoquinone
showed that the relative intensities of the protonated and
cationized molecules may distinguish between these
compounds, without the addition of acid. The same conclu-
sions can be applied to a- and b-lapachone. ESI-MS/MS
analyses of protonated 1,2- and 1,4-naphthoquinones have
shown that the relative intensity and number of product ions
helped characterize these compounds. These results will
serve to identify these compounds in complex mixtures
or metabolism studies. The fragmentation mechanisms for
a- and b-lapachones are very similar. The most intense
product ion stems from a RDA reaction, and the different
relative intensities of ions during CID experiments distin-
guish these protonated molecules from their isomers, such
as lapachol and isolapachol. Computational results corrobo-
rated the ESI-MS and MS/MS data, and all the results can be
used in other studies involving naphthoquinones.
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SUPPORTING INFORMATION
Supporting information may be found in the online version of
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Acknowledgements
The authors thank the Brazilian foundations FAPESP, CAPES,
INCT _ if and CNPq for financial support. Ricardo Vessecchi
thanks FAPESP and CAPES/PNPD for a postdoctoral scholarship.
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