Gas-phase reactivity of 2-hydroxy-1,4-naphthoquinones: A computational and mass spectrometry study of lapachol congeners

Article abstract of DOI:10.1002/jms.3101

In order to understand the influence of alkyl side chains on the gas-phase reactivity of 1,4-naphthoquinone derivatives, some 2-hydroxy-1,4-naphthoquinone derivatives have been prepared and studied by electrospray ionization tandem mass spectrometry in combination with computational quantum chemistry calculations. Protonation and deprotonation sites were suggested on the basis of gas-phase basicity, proton affinity, gas-phase acidity (?G _acid), atomic charges and frontier orbital analyses. The nature of the intramolecular interaction as well as of the hydrogen bond in the systems was investigated by the atoms-in-molecules theory and the natural bond orbital analysis. The results were compared with data published for lapachol (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone). For the protonated molecules, water elimination was verified to occur at lower proportion when compared with side chain elimination, as evidenced in earlier studies on lapachol. The side chain at position C(3) was found to play important roles in the fragmentation mechanisms of these compounds.

Full text of DOI:10.1002/jms.3101

Research article
Received: 24 May 2012
Revised: 3 September 2012
Accepted: 4 September 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3101
Gas-phase reactivity of 2-hydroxy-1,4-
naphthoquinones: a computational and mass
spectrometry study of lapachol congeners
a
b
c
Ricardo Vessecchi, * Flávio S. Emery, Sérgio E. Galembeck and
a
Norberto P. Lopes *
In order to understand the influence of alkyl side chains on the gas-phase reactivity of 1,4-naphthoquinone derivatives, some
2-hydroxy-1,4-naphthoquinone derivatives have been prepared and studied by electrospray ionization tandem mass
spectrometry in combination with computational quantum chemistry calculations. Protonation and deprotonation sites were
suggested on the basis of gas-phase basicity, proton affinity, gas-phase acidity (ΔGacid), atomic charges and frontier orbital
analyses. The nature of the intramolecular interaction as well as of the hydrogen bond in the systems was investigated by
the atoms-in-molecules theory and the natural bond orbital analysis. The results were compared with data published for
lapachol (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone). For the protonated molecules, water elimination was
verified to occur at lower proportion when compared with side chain elimination, as evidenced in earlier studies on lapachol.
The side chain at position C(3) was found to play important roles in the fragmentation mechanisms of these compounds.
Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: quinone; gas-phase ion chemistry; proton affinity; theoretical calculations; fragmentation studies; even-electron rule
compounds exhibiting different side chains and which can be
Introduction
obtained from natural sources, biomimetic reactions or synthetic
Quinonoid compounds are an important class of ubiquitous
[
1–4]
routes. Because products can be generated at low concentration,
it is crucial that a highly sensitive technique be used for their
structural elucidation.
natural products with action against several tropical diseases.
In South America, some plant species have been shown to display
[
5,6]
2-hydroxy-1,4-naphthoquinones as taxonomic markers.
More-
Knowing that the side chain of quinonoid compounds has
a relevant effect on the fragmentation and reactivity of 2-
hydroxy-1,4-naphthoquinone derivatives during ESI-MS/MS
analysis, in this work, we have conducted ESI-MS/MS studies in
the positive and negative modes, in order to produce protonated
and deprotonated molecules. We have used a combination of
DFT calculations with the B3LYP/6-31 + G(d,p) computational
over, the naturally-occurring lawsone (Q1), isolapachol (Q3) and
lapachol (Q4) (Fig. 1) have been demonstrated to possess antitu-
moral and antiparasitic activities.
behavior of this class of compounds is interesting, since their action
mechanism is related to radical or hydroquinone species.
this reason, the synthesis of new quinonoid compounds has
become an attractive source of bioactive compounds.
Many researchers have devoted their studies to the prepara-
tion and rapid characterization of quinonoids.
electrospray ionization tandem mass spectrometry (ESI-MS/MS)
has been increasingly utilized for the structural elucidation
and characterization of new natural compounds,
[
7–10]
The reduction/oxidation
[
11,12]
For
[
13–15]
[
22,23]
[24]
model
and energy-resolved plots to suggest the collision-
[
16]
induced dissociation (CID) processes. Protonation and deprotona-
tion sites have been hypothesized on the basis of the atomic
In this sense,
[
17]
and this
technique has recently been applied to the analysis of routes
[
18,19]
* Correspondence to: Ricardo Vessecchi and Norberto P. Lopes, Núcleo de
Pesquisa em Produtos Naturais e Sintéticos, 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. E-mail:
vessecchi@bol.com.br, vessecchi@usp.br, npelopes@fcfrp.usp.br
for the synthesis of natural products.
Gas-phase chemistry
studies in combination with computational chemistry of
derivatives and congeners have also become an important tool
for the characterization and structural elucidation of novel
[20,21]
substances.
a 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
In this context, our research group has concentrated efforts on
the investigation of lapachol (Q4) by means of high-resolution
ESI-MS/MS experiments in combination with computational
[16]
b Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas
de Ribeirão Preto, Universidade de São Paulo, Brasil
chemistry.
We have evaluated the protonated, deprotonated
and cationized molecules, and we have elucidated the fragmen-
tation mechanism on the basis of electronic structure calcula-
tions. These results have aided the characterization of new
c Departamento de Química, Faculdade de Filosofia Ciências e Letras de
Ribeirão Preto, Universidade de São Paulo, Brasil
J. Mass Spectrom. 2012, 47, 1648–1659
Copyright © 2012 John Wiley & Sons, Ltd.

Gas-phase chemistry of hydroxy-quinonoid compounds
O
O
(
1)
charges, frontier orbitals, proton affinities and gas-phase acidities,
among other quantum chemistry reactivity descriptors.
2)OH
OH
(1)
(
(4) (3)
O
O
(
2)
Experimental
Q1
Q2
Synthesis and MS
O
O
Lawsone (Q1) was commercially available from Aldrich. Dihydro-
lapachol (Q2) was prepared by catalytic hydrogenation of
OH
OH
[
25]
lapachol (Q4) with PtO2
(Scheme 1). Isolapachol (Q3) was
synthesized from Q1 in moderate yields, by condensation of
O
Q3
O
Q4
[
26,27]
isovaleraldehyde in acidic media
(Scheme 2).
Synthesis of isolapachol (Q3): Concentrated HCl (2.0 ml) and
isovaleraldehyde (5.0 ml) were added to a boiling solution of
Q1 (2.0 g, 11,5 mmol) in glacial acetic acid (35 ml). The reaction
Figure 1. Structure of 2-hydroxy-1,4-naphthoquinone derivatives. Q1
lawsone, Q2 dihydrolapachol, Q3 isolapachol and Q4 lapachol. The
numbered atoms indicate the position on the quinone moiety.
ꢀ
mixture was stirred at 80 C for 2.0 h and poured into water
(200 ml). The solution was left to stand overnight, an oily residue
separated, and this residue was dissolved in ether (100 ml). The
ethereal solution was extracted with NaOH 1% (two 200 ml
portions). The violet basic aqueous solution was acidified
O
O
O
O
OH
OH
H2, 1 atm, PtO2,
AcOEt, 5h, 77%
Scheme 1. Preparation of dihydrolapachol (Q2).
O
O
OH
OH isovaleraldehyde, HCl,
AcOH, 80° C, 2h, 46.7%
O
O
Scheme 2. Preparation of isolapachol (Q3).
O
H
O
O
O
H
O
0.0)
O
(6.67)
(
O
H
O
O
O
H
O
(6.30)
O
(
0.0)
O
H
O
O
O
H
O
(5.69)
O
(
0.0)
Figure 2. Rotational isomers of 2-hydroxy-1,4-naphthoquinone derivatives.
ꢁ
1
Values are the relative Gibbs energies, in kcal.mol , obtained at the B3LYP/
-31+G(d,p) level.
Figure 3. Molecular graphs for neutral compounds obtained by AIM
analysis.
6
J. Mass Spectrom. 2012, 47, 1648–1659
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

R. Vessecchi et al.
(
conc. HCl), and the solid residue was consecutively recrystallized in
constructed, for better understanding of the dissociation processes
undergone by the protonated and deprotonated molecules.
petroleum ether (35–55º) and ethanol, which furnished orange iso-
lapachol crystals (1.3g, 46.7%) (Scheme 2). The synthesis of Q2 and
Q3 was confirmed by spectroscopy, and the results were compared
with literature data.
Computational methods
All the molecules were optimized by means of the B3LYP/
Q1, Q2 and Q3 stock solutions were prepared for MS analysis
from a 0.1 mg. ml solution of each compound in acetonitrile/
water (2:1 v/v).
The high-resolution ESI-MS analyses were accomplished on an
UltroTOF-Q apparatus (Bruker Daltonics, USA) fitted with an
electrospray ion source operating in the positive and negative
ion modes. The accuracy masses were obtained by using
sodiated trifluoroacetic acid as mass standard, and the molecular
formulae were suggested on the basis of the errors lower than
[
22,23]
[28]
ꢁ
1
6-31 + G(d,p) model,
using Gaussian 03 suite programs.
Geometry analysis for 2-hydroxy-1,4-naphthoquinone has shown
that the bond length values agree with X-ray diffraction experi-
[
29]
ments.
In a thermochemical study of models of natural
carbonyl compounds, this was the model that produced the
[
30]
closest results to the experimental data
Recently, we have
demonstrated that the B3LYP model is the most exact functional
for calculation of the thermochemical parameters of quinonoid
[
31]
compounds.
the structures are minima in the potential energy surface. The
The vibrational frequencies indicated that all
5
ppm. Samples were directly infused into the ionization source
ꢁ
1
at a flow rate of 10 ml.min . The source block and desolvation
[
32,33]
ꢀ
Molekel 4.3 and JMol 9.0 softwares
were employed for
temperature was 150 C. The optimum energy applied at the
visualization of some of the examined properties. natural bond
orbital (NBO) analyses were carried out by using the NBO 5.0
software interfaced with the Gaussian 09 suite program.
capillary emitter was tested, in order to achieve a larger intensity
of protonated and deprotonated molecules.
[
34]
[35]
The precursor ion was selected and fragmented by CID, using N
as collision gas at a specific Elab value Energy-resolved plots were
2
Protonation sites were achieved on the basis of HOMO
analysis, molecular electrostatic potential map (MEP), natural
charges, the relative Gibbs energies between several protonated
.
2
Table 1. Values of r, r r, and H
b
for BCPs of HBs as obtained by AIM
[
36]
forms (gas-phase basicity (GB)) and proton affinities (PA).
analysis, in a.u.
Deprotonation sites were obtained by comparing the natural
charges of hydrogen atoms and gas-phase acidity (ΔGacid) values.
The ΔGacid value of each molecule was calculated by subtract-
ing the thermally corrected Gibbs energy of the acid from the
sum of the thermally corrected Gibbs energies of the anion and
2
Molecule
r
r r
H
b
Q1
Q2
Q3
0.026
0.030
0.027
0.09
0.10
0.09
0.0004
0.0004
0.0000
proton. The ΔG
values were computed according to the
acid
-
+
equation: QH ! Q + H , where the Gibbs energy of the proton
ꢁ
1 [37]
2
was considered as being ꢁ6.28 kcal.mol
.
The GB values were
Table 2. Energy of second-order interactions (ΔE ) for HB, in kcal.
ꢁ
1
mol
obtained from variations of the Gibbs energies for a protonation
+
+
reaction Q + H ! QH .
Molecules
[
38]
The quantum theory of atoms-in-molecules (AIM)
analysis
NBO
interaction
Q1
syn
Q1
anti
Q2
syn
Q2
anti
Q3
syn
Q3
anti
was applied, so as to gain insight into the changes in bond
strength after a chemical event, such as protonation or
[
39]
deprotonation.
the aid of the AIM 2000 software.
The molecular graphs were plotted with
n
n
pO3 ⇒ p*C2-C3
pO1 ⇒ s*O3-H1
38.03 36.57 34.68 35.47 34.94 34.74
[
40]
4.75
6.41
0.88
2.08
–
–
–
–
5.44
6.25
0.83
1.98
–
–
–
–
5.09
6.26
0.77
2.08
–
–
–
–
For the fragmentation mechanism studies, the most stable ions
were proposed, and the fragmentation pathways were suggested
by Gibbs energies and enthalpies calculations at 298.15 K. The pro-
ducts and the corresponding transition state (TS) energies were
sO3-H1 ⇒ s*C2-C3
sO3-H1 ⇒ s*C1-C2
sC2-C3 ⇒ s*O3-H1
Figure 4. HOMO and MEP for Q1, Q2 and Q3, respectively.
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J. Mass Spectrom. 2012, 47, 1648–1659

Gas-phase chemistry of hydroxy-quinonoid compounds
obtained and reported relative to their respective precursors. All
O
H
O
O
H
O
O
H
O
the TS were found by means of Synchronous Transit Guided
Quasi-Newton method developed by Schlegel and co-workers,
[
41]
and were characterized by one imaginary vibrational frequency.
O
O
O
Q1
Q2
Q3
Results and discussions
H+
H+
H+
Protonation/deprotonation sites
H
H
H
Recent studies with 1,4-naphthoquinone derivatives have shown
that the occurrence of a radical anions during ESI can be related
O
O
O
O
O
O
[
31,42]
H
H
H
to the reduction potential, ΔGacid and electron affinity.
Here,
no radical anions/cations were detected during the MS analysis.
Thus, we concluded that acid–base reactions take place in high
proportion for 2-hydroxy-1,4-naphthoquinone derivatives, which
generates the protonated and deprotonated molecules.
O
O
O
(
192.49)
(199.61)
192.63
(201.49)
194.46
1
85.36
+
+
+
Q1H _1
Q2H _1
Q3H _1
To understand the ESI-MS investigations, the first step is to
gain knowledge about the reactivity of neutral, protonated
[
36,39]
and deprotonated species.
The neutral 2-hydroxy-1,
H
O
H
O
H
O
4-naphthoquinone exhibits two most stable forms, which can
be related to O–H bond rotation. Energy analyses indicated
that the hydrogen atom is parallel to the carbonyl at position
C(1). These results are in agreement with those described by
H
O
H
O
H
O
[
43]
Lamoureux et al.
for this same compound, for which syn-
O
O
O
hydroxyl is the most stable tautomer.
(
199.28)
85.23
Q1H _2
(200.23)
193.11
(207.04)
200.05
Q3H _2
The stability of the syn-hydroxyl molecules (Fig. 2) can be
attributed to the intramolecular hydrogen bond (HB) that can
be established between O(1) and H–O(3), as evidenced by the
AIM analyses (Fig. 3), which also revealed the presence of a bond
1
+
+
+
Q2H _2
H
O
H
O
[44]
2
H
O
path between these atoms.
Analysis of r and r r for the
O
O
bond critical point (BCP) of these bonds suggested that these
O
H
H
H
interactions are indeed HBs, as judged from the low r value
2
[44]
and positive r r value,
and bearing in mind that an HB has
2
[45]
O
O
0.01 < r < 0.04 and 0.04 < r r < 0.12 (Table 1).
Another
O
(
207.13)
00.01
(
196.65)
92.22
Q1H _3
(208.17)
200.68
Q3H _3
parameter that indicated the occurrence of an HB was the
2
1
Hamiltonian total energy density (H ). This parameter was
b
+
+
Q2H _3
+
calculated from the relative magnitudes of kinetic energy densities
[
45]
(
G ) and potential energy densities (V ), where G + V = H . Hb
b b b b b
values ranging from ꢁ0.004 to 0.002a.u. suggested the presence
O
O
O
of an HB (Table 1).
O
O
O
H
H
H
These HBs were also characterized by NBO analysis. The
stability of the syn-hydroxyl form was evident from the energy
of second-order interactions between formally occupied and
O
O
H
O
H
H
2 [46]
formally empty NBOs (ΔE ).
When the same interactions
(
200.86)
93.66
Q1H _4
(204.08)
197.03
(206.51)
199.66
were compared for Q1, e.g. n ⇒ p*C2-C3, the syn conformer
pO3
ꢁ1
1
2
presented ΔE = 38.03 kcal.mol , while for the anti conformer,
+
+
+
Q2H _4
Q3H _4
2
ꢁ1
this interaction had ΔE = 36.57 kcal.mol . Nevertheless, other
interactions indicating the existence of an HB could be verified,
and overall interactions contributed to the stabilization of the
O
O
H
O
H
O
O
O
H
2
ꢁ1
syn form npO1 ⇒ s*O3-H1, ΔE = 4.75 kcal.mol ; sO3-H1 ⇒ s*C2-C3,
O
O
O
H
(211.20)
203.83
O
O
O
O
H
H
OH
R
O
R
(
211.52)
(212.43)
205.55
H+
-
2
08.66
+
+
+
Q1H _5
Q2H _5
Q3H _5
Q1 (312.83)
Q2 (313.09)
Q3 (311.33)
Scheme 3. Proton affinities (between parenthesis) and gas-phase
basicities (italic values) calculated at the B3LYP/6-31 + G(d,p) level. All
ꢁ
1
the values are given in kcal.mol . Squares indicate the most stable
cation. All geometries and absolute values are presented in the
supporting information.
Scheme 4. Gas-phase acidity of 2-hydroxy-1,4-naphthoquinone deriva-
1
ꢁ
tives. All the values are given in kcal.mol and were calculated at
B3LYP/6-31 + G(d,p) level.
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R. Vessecchi et al.
2
ꢁ1
2
ꢁ1
Table 3. Accurate mass values observed during ESI-MS analysis of
the target compounds, Fig. 1
ΔE = 6.41 kcal.mol
;
sO3-H1 ⇒ s*C1-C2
,
ΔE = 0.88 kcal.mol
2
ꢁ1
and sC2-C3 ⇒ s*O3-H1, ΔE = 2.08 kcal.mol . All these interactions
were noted for Q2 and Q3 in the same proportions (Table 2).
For location of the most favorable protonation site in the target
compounds, we considered the syn and anti isomers. The HOMO
at the equilibrium geometries of the compounds was examined
Fig. 4), to obtain guidance about the most basic site in the
molecules. HOMO was found to be localized between the O(2), O
3), C(2) and C(3) atoms, which suggests preferential proton
Molecular
formulae
Exact
Mass
Observed
Mass
Error
(ppm)
+
+
+
+
[Q1 + H]
[Q2 + H]
[Q3 + H]
C
10
C
15
C
15
H
H
H
7
O
3
175.03897
245.11722
243.10157
175.0389
245.1172
243.1016
0.4
0.1
0.1
+
17
15
O
3
(
+
3
O
(
attachment to these atoms. The MEP was also constructed for each
compound, and the most nucleophilic regions were found to be
the oxygen atoms on the quinonoid moiety (Fig. 4). We have
used these parameters to obtain the protonation sites for polyfunc-
Intens.
1
47.0400
[
17,20,21]
1
1
1
500 A
tionalized molecules, and the results were satisfactory.
In order to understand the effects of protonation on the
energies and geometries of these compounds, all the basic sites
were examined, as depicted in Scheme 3.
250
000
1
05.0317
7
5
2
50
00
50
0
(A)
1
1
1
00
m/z 175
m/z 147
m/z 129
m/z 105
m/z 77
80
1
75.0319
m/z 69
68.9995
60
1
29.0320
7
7.0412
4
2
0
0
0
6
0
80
100 120 140 160 180 m/z
Intens.
1
89.0547
B
-
5
0
5
10
15
Elab (eV)
20
25
30
35
35
40
40
6
4
2
000
000
000
0
(B)
m/z 245
m/z 227
m/z 209
m/z 189
m/z 171
m/z 159
m/z 143
m/z 115
00
80
1
43.0489
2
09.0957
1
71.0482
60
2
45.1163
40
2
27.1056
1
15.0524
20
0
1
00 120 140 160 180 200 220 240 m/z
Intens.
-5
0
5
10
15
Elab (eV)
20
25
30
40
1
87.0391
1
1
1
500 C
2
43.1013
(C)
00
m/z 243
m/z 225
m/z 201
m/z 187
m/z 159
m/z 131
m/z 103
250
8
6
4
0
0
0
000
7
5
2
50
00
50
0
2
25.0906
20
0
159.0406
201.0585
0
10
20
30
Elab (eV)
1
00 120 140 160 180 200 220 240 m/z
+
Figure 6. Energy-resolved plots for the dissociation of [Q + H] ions.
+
+
+
Figure 5. ESI-MS/MS spectra for 2-hydroxy-1,4-naphthoquinone deriva-
tives at Elab = 15 eV. A) [Q1 + H] , B) [Q2 + H] and C) [Q3 + H] .
(A) [Q1 + H] , (B) [Q2 + H] and (C) [Q3 + H] . All the spectra can be
assessed at supporting information, Figs. S7, S9 and S11.
+
+
+
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J. Mass Spectrom. 2012, 47, 1648–1659

Gas-phase chemistry of hydroxy-quinonoid compounds
The protonation at OH led to a most unstable ion for Q1, Q2
and Q3, because the proton migrated to O(1) during geometry
MS and fragmentation mechanisms
+
+
Positive ions
optimization (see equilibrium the structure Q1H _3, Q2H _3,
+
Q3H _3 in Scheme 3). The most stable ion was generated upon
Accurate mass analyses were conducted, and the molecular
formulae of the expected ions were elucidated on the basis of
the obtained ppm error (Table 3). All the ESI-MS spectra are
illustrated in the supporting information (Figs. S1, S3 and, S5).
The protonated molecules were selected and dissociated by
collision (ESI-MS/MS experiments), and the results will be discussed
in the next section (Fig. 5).
+
+
protonation of atom O(2), which gave Q1H _5, Q2H _5, and
+
Q3H _5, as shown in Scheme 3. Protonations at C(2) and C(3)
atoms should be less energetic than those protonated at O
atoms, for example, for Q1 protonated at C(2), the PA value is
ꢁ
1
1
80.75 kcal.mol . Similar results were obtained to protonation
at vinyl carbons of Q3. Isolapachol (Q3) was found to be the
most basic compound and, the higher difference between GB
when compared to PA values can be attributed to structural
and entropy effects by presence of unsaturated lateral chain, as
can be observed at Fig. 3.
Water loss was not observed for the protonated 2-hydroxy-1,
+
4-naphthoquinone molecule [Q1 + H] irrespective of the applied
Elab (Fig. 6(A)). CO elimination was the most important frag-
mentation channel for the protonated molecule, and water
elimination happened after this process, affording the m/z
129 ion, Figs. 6(A) and S7. This behavior was similar to that
exhibited by the radical cation of 1,4-naphthoquinone and
Deprotonation sites were easily evidenced by analyses of the
atomic charges and acidity (Scheme 4), as previously described
[17]
for Q4.
Deprotonation occurred at the hydroxyl hydrogen
[
47]
atom, furnishing to an enolate-type ion that could be stabilized
2-hydroxy-1,4-naphthoquinone upon EI-MS analysis.
Ketene
[17]
by resonance.
Q3 was found to be the most acidic compound, with ΔGacid
loss from the ion of m/z 147 may furnish the m/z 105 ion.
For this reason, the MS/MS of m/z 147 was obtained. For the
=
ꢁ
1
+
+
+
3
11.33 kcal.mol . This can be explained by the resonance
[Q2 + H] , [Q3 + H] , and [Q4 + H] fragmentation, however, water
loss occurred before CO elimination, and the m/z 225, 227 and
248 ions were detected in the mass spectra (Fig. 6) as reported
between the enolate form and the unsaturated side chain, which
extended the conjugation.
O
O
O
O
OH
O
OH
OH
OH
OH
I)
(
X
b)
H
(
-CO
OH
OH
OH
OH
m/z 147
4.71)
(15.80)
OH
TS
(64.93)
76.51)
m/z 147
m/z 175
0.0)
m/z 175
TS
(48.92)
50.96)
(5.83)
(
(
0.0)
(17.95)
(
(
(
(
II)
(
a)
O
O
OH
+
H O
2
O
OH
27.69)
40.14)
O
21.70)
22.27)
(
(
m/z 129
(41.73)
55.41)
(
(
(
O
O
H
+
CO
O
O
H
m/z 101
(
26.38)
(40.05)
O
+
C H
8
6
H
H
OH
CH CO +
2
O
OH
O
O
O
OH
+
m/z105
O
O
O
m/z 105
(68.02)
(83.32)
(
(
21.95)
23.37)
(49.80)
(52.77)
+
Scheme 5. Fragmentation mechanisms for [Q1 + H] . Values between parentheses are relative Gibbs energies at 298 K. Italic values are relative
ꢁ
1
enthalpies at 298 K. All the values are expressed in kcal.mol and were computed at the B3LYP/6-31 + G(d,p) level.
J. Mass Spectrom. 2012, 47, 1648–1659
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

R. Vessecchi et al.
[
17]
in our early studies on Q4.
Water elimination was more pro-
The higher intensity of this ion was verified at Elab > 20 eV. Other
TS were obtained, in order to explain the occurrence of m/z 105,
which should be due to ring opening and ketene loss (pathway (b)).
The TS relative critical energy and the products energies were
very close.
+
+
+
nounced for [Q4 + H] , comparedwith[Q2+H] and [Q3 + H] (Fig. 6).
This can be attributed to the most stable protonated form, which
is different for [Q4 + H] as compared to [Q2 + H] and [Q3 + H] .
The fragmentation mechanism for the formation of m/z 147
from [Q1 + H] can be seen in Scheme 5 (pathway I). The
generation of m/z 147 occurred via ring contraction through CO
elimination. The critical Gibbs energy for this step was obtained
as being 48.98 kcal.mol (TS1, Scheme 5). The relative Gibbs
energy and enthalpy for this step were calculated as being 4.71
and 15.8 kcal.mol , respectively, which indicate the higher
intensity of this ion. We can take into account, for instance, the
+
+
+ [17]
+
In order to confirm the fragmentation channel to generate
m/z 105, the MS/MS analyses of m/z 147 were obtained, and
the results can be assessed at supporting information (Fig. S13).
The fragmentation of m/z 147 does not generate the m/z 105 ion.
ꢁ
1
+
Thus, we conclude that the m/z 105 is occurring by [Q1 + H]
ꢁ
1
fragmentation and, the pathway (b) can be discarded. The
fragment m/z 105 should be produced via ring opening, going
through multiple steps mechanism, as depicted at Scheme 5,
pathway II. Moreover, the experimental data confirm that the
pathway I(a), Scheme 5, as the most probable channel for
fragmentation of m/z 147 and formation of m/z 129 and m/z 101
ions. The fragment m/z 129 (pathway (a)) should be generated
via endothermic hydrogen migration, in a similar way to the
E
lab of 5 eV. The maximum energy transfer (ECM) from simple
ꢁ
1
collision with N
2
1 [48]
was 15.9 kcal.mol
Thus, this ion would be produced from
eV, as can be observed in Fig. 6(A).
Two fragmentation processes may have taken place from
the resulting m/z 147 ion (pathways (a) and (b)). First, water
loss may have yielded to the m/z 129 ion, as represented in
Scheme 5- pathway (a). Second, neutral ketene elimination might
have culminated in m/z 105 (Scheme 5-pathway (b)). Analysis of
the energy-resolved plot for [Q1 + H] evidenced that m/z 105
formation should be easier than m/z 129 generation (Fig. 6-A).
The fragment m/z 147 should be produced via hydrogen
([28/(175 + 28)]x 5 eV =
ꢁ
1
5.9 kcal.mol ).
5
[
49]
Grob fragmentation.
+
For [Q2 + H] (m/z 245), the most intense fragment ion was
m/z 189, which can be ascribed to 2-methyl-propene side chain
elimination (Fig. 5(B), 6(B), S9 and Scheme 6). Considering that
protonation occurred at O(2), the Gibbs energy for this process
+
ꢁ
1
was calculated as being ꢁ2.33 kcal.mol . The relative enthalpy
ꢁ
1
ꢁ1
migration, going through a TS with 76 kcal.mol , Scheme 5.
necessary for this elimination was approximately 10 kcal.mol
.
O
O
OH
OH
OH
+ C H
H
4
8
+ CO
OH
OH
OH
+
Q2H _5
(
m/z 189
(-2.33)
(10.10)
m/z 161
(2.04)
(26.62)
0.0)
OH
OH
O
OH
OH
OH
+
2
-H O
H
+ CO
OH
C4H8
O
O
m/z 143
(
10.66)
+
Q2H _3
3.82)
4.10)
m/z 189
4.81)
17.78)
(35.43)
-CO
(
(
m/z 161
(
(
O
-CO
+
CO
O
O
m/z 143
m/z 115
m/z 171
C9H7+
O
O
O
O
-CO
-CO
O
O
m/z 143
m/z 171
m/z 143+
C10H7O
+
m/z 171
C10H7O
(
(
42.96)
69.13)
+
Scheme 6. Fragmentation mechanisms for [Q2 + H] . Values between parentheses are relative Gibbs energies at 298 K. Italic values are relative
ꢁ
1
enthalpies at 298 K. All the values are expressed in kcal.mol and were computed at the B3LYP/6-31 + G(d,p) level.
wileyonlinelibrary.com/journal/jms
Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2012, 47, 1648–1659

Gas-phase chemistry of hydroxy-quinonoid compounds
+
+ [17]
Thus, protonation may be considered the starting point of the
fragmentation pathway. Water loss from the two most stable
protonated ions [Q2 + H] should happen by means of an
endothermic process. The fact that m/z 227 had the lowest
intensity in the ESI-MS/MS spectra could be due to vinyl
cation formation, which could sustain rearrangement to a five-
membered ring. More intense m/z 171 and 143 peaks were
verified after 15 eV. The formation of these ions should take place
via side chain elimination, to produce m/z 189, which would
[Q2 + H] and [Q4 + H] . According to the proposed mechanism,
+
[Q3 + H] should be formed by tautomerization of Q3 to Q4. The
+
critical energy for this conversion was calculated as being
12.02 kcal.mol . The relative energy between these two
ꢁ
1
Table 4. Accurate mass values obtained by ESI-MS (negative mode)
analysis of the compounds, Fig. 1
Molecular Exact mass Observed
Absolute error
(ppm)
2
undergo subsequent H O and CO losses. Several steps were
formulae
mass
necessary for m/z 171 formation from m/z 245. The relative
-
-
-
-
ꢁ
1
[Q1-H]
[Q2-H]
C10H5O
173.02442
243.10267
241.08702
173.0244
243.1028
241.0874
0.1
0.5
1.6
3
enthalpy determinated for m/z 171 was 36.8 kcal.mol , and this
-
ꢁ
1
C15H15
O
O
3
value was very close to that obtained for ECM; i.e. 35.3 kcal.mol
.
-
[
Q3-H]
C
15
H
13
3
Generation of fragment m/z 143 from m/z 171 could be triggered
by two alternative pathways, namely CO elimination through ring
contraction and ring opening followed by CO loss and phenyl
cation formation, Scheme 6.
+
(A)
[
Q3 + H] fragmentation was similar to that described for
100
+
[17]
lapachol [Q4 + H] .
The most intense ions in the ESI-MS/MS
m/z 173
m/z 145
m/z 101
spectra were m/z 187 and m/z 159 (Fig. 5(C), 6(C) and S11).
However, water elimination occurred at low energies (0ꢁ20 eV),
and this process happened to a less extent as compared to
8
6
4
0
0
0
O
H
O
O
(
12.02)
O
(
7.80)
20
0
H
H
O
O
H
H
0
10
20
Elab (eV)
30
30
20
40
(
0.0)
(5.53)
(5.14)
m/z 243
(
B)
1
00
80
m/z 243
m/z 215
m/z 200
m/z 199
m/z 186
m/z 171
O
H
O
O
O
H
+
6
4
2
0
0
0
0
O
H
O
H
m/z 243
m/z 187
(
28.25)
(
40.93)
O
O
0
10
20
Elab (eV)
40
+
H2O
+
CO
(
C)
O
1
00
OH
m/z 159
m/z 241
m/z 226
m/z 213
m/z 198
m/z 186
80
60
(
(
11.68)
35.39)
O
4
2
0
0
0
+
H2O
O
m/z 225
(
(
28.45)
41.19)
0
5
10
15
Elab (eV)
25
30
+
Scheme 7. Fragmentation mechanisms for [Q3 + H] . Values between
-
parentheses are relative Gibbs energies at 298 K. Italic values are relative
enthalpies at 298 K. All the values are expressed in kcal.mol and were
computed at the B3LYP/6-31 + G(d,p) level.
Figure 7. Energy-resolved plots for deprotonated molecules. (A) [Q1-H] ,
ꢁ1
-
-
(B) [Q2-H] and (C) [Q3-H] . All the spectra can be assessed at supporting
information, Figs. S8, S10 and S12.
J. Mass Spectrom. 2012, 47, 1648–1659
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

R. Vessecchi et al.
ꢁ
1
ꢁ1
conformers was 5.53 kcal.mol . Side chain elimination might
take place via a sygmatropic rearrangement through pericyclic
reaction, to produce m/z 187 (Scheme 7). The latter fragment
could eliminate a CO molecule and generate m/z 159 by ring
contraction, through an exothermic step (ΔH298 = ꢁ5 kcal.mol ).
AIM studies have shown that Q4 protonation can promote a
All the mechanisms for m/z 159 and
m/z 187 formation have been elucidated during ESI-MS/MS
[
17]
pericyclic reaction.
O
O
O
O
O
O
O
m/z 173
[
m/z 145_b
Q4-H]-
(2.16)
(
13.30)
H
O
O
O
O
O
O
O
m/z 101
m/z 145_d
(50.90)
m/z 145_e
(37.62)
m/z 145_a
O
(
76.61)
98.79)
m/z 145_c
(
(
16.17)
26.95)
(
(
62.12)
(
51.38)
-
Scheme 8. Fragmentation mechanism for [Q1-H] . Values are relative Gibbs energies, calculated at the B3LYP/6-31 + G(d,p) level. Italic numbers are
ꢁ1
relative enthalpies at 298 K. All the values are in kcal.mol
.
O
O
O
O
O
O
m/z 243
(
m/z 186
45.86)
59.44)
0.0)
(
(
O
O
O
O
O
O
m/z 215_a
(-0.59)
m/z 215_TS
(
135.21)
(-12.09)
(
134.84)
-
CH3
m/z 215_b
(
(
17.22)
28.53)
O
-
CH3
O
O
m/z 200_a
CO2
O
(
(
63.48)
66.12)
m/z 215_c
32.29)
23.64)
(
(
m/z 200_b
79.28)
104.99)
(
(
m/z 171
(
72.25)
(
74.46)
-
Scheme 9. Fragmentation mechanism for the [Q2-H] ion. Values are relative Gibbs energies at 298 K, calculated at the B3LYP/6-31 + G(d,p) level. Italic
numbers are relative enthalpies.
wileyonlinelibrary.com/journal/jms
Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2012, 47, 1648–1659

Gas-phase chemistry of hydroxy-quinonoid compounds
[
50]
investigations involving lapachol. The TS of the pericyclic
reaction was also obtained by the same computational model,
and these results could be exploited.
isoflavone.
Considering the maximum energy transferred
through collision between N2 and the m/z 145 ion, which is
[
17]
ꢁ1
96 kcal.mol , it can be inferred that this ion should occur at Elab
higher than 30eV (Fig. 7(A)).
-
[
Q2-H] dissociation gave rise to three major ions (m/z 215,
Negative ions
m/z 200 and m/z 186) in the ESI-MS/MS spectrum (Figs. 7(B) and S10).
[
51,52]
High-resolution ESI-MS analysis of 1,4-naphthoquinone deriva-
tives in the negative mode showed that the most intense ions
corresponded to the deprotonated molecules (supporting
information, Figs. S2, S4 and S6). The molecular formula for
each ion was identified by examination of the absolute errors
On the basis of recent publications,
which have described
that exception to the even-electron rule occurs for negative ions
during CID experiments, m/z 186 could be suggested. Indeed,
this fragment has been detected in studies on deprotonated
[
17,52]
naphthoquinones,
where the most abundant fragment ions
(
Table 4).
appeared due to exception to the even-electron rule. In our case,
m/z 186 formation was probably a result of the homolytic
cleavage of the side chain via elimination of a t-butyl radical
(Scheme 9). The computed enthalpy for this reaction was
59.43kcal.mol . This step involved higher energy than CO loss
(m/z 243 ! m/z 215), which indicates that m/z 186 was generated
after m/z 215 formation (Scheme 9).
On the basis of earlier studies with Q4, the deprotonated site
-
was suggested as being the hydroxyl. The [Q-H] ions were
submitted to CID conditions, and the energy-resolved plots were
obtained, as illustrated in Fig. 7. The fragmentation mechanism
for each compound was proposed from these results.
ꢁ
1
-
The [Q1-H] fragmentation mechanism started by formation of
m/z 145, the most intense fragment ion in the mass spectrum
Initiation of m/z 200 ion formation takes place via homolytic
(
[
Figs. 7(A) and S8). This ion can be attributed to CO loss from
Q1-H] , with consequent ring contraction. Two pathways can
cleavage of the side chain, with methyl radical (
•
3
CH ) elimination.
-
Radical losses from open-shell ions have been observed in ESI
[
17,51,52]
be proposed for this CO elimination, by considering the quino-
studies.
The lower intensity of m/z 200 can be explained
noid carbonyl system. First, the CO loss would occur at C(2)–O
by enthalpy values (ΔH298 = 66.12, for m/z 200_a and ΔH298
=
(
3) to afford m/z 145_a (Scheme 8). Alternatively, CO elimination
104.99 for m/z 200_b). The fragment m/z 171 should be pro-
duced via a mechanism similar to the one reported for m/z 101
generation from [Q1-H] (Scheme 8). Formation of these ions
could take place at C(1)–O(1), to form m/z 145, which would be
stabilized through resonance (m/z 145_b and m/z 145_c,
Scheme 8). The possible resonance can explain the intensity of
-
should depend on the keto-enol intermediate, for which ΔH298,
-
ꢁ1
this ion in the MS/MS spectra of [Q1-H] , Fig. 7(A). The relative
TS = 134.84 kcal.mol
.
-
enthalpy for m/z 145 formation from m/z 173 (m/z 173 ! m/z
[Q3-H] dissociation should be similar to that described for Q4,
ꢁ
1
1
45_b) was calculated as being 13.30 kcal.mol , indicating the
and the most intense fragment ion should be m/z 186 (Fig. 7(C)
[
17]
occurrence of this in low energies processes. For m/z 101
production, it would be necessary that m/z 145_b went
through a TS, as demonstrated in studies involving isoflavone
and S12).
Other ions were observed in lower proportion.
The iso-lapacholate ion might rearrange to its tautomer, the
lapacholate ion. The critical energy for this step was determined
[50]
ꢁ1
negative ions,
to eliminate CO . The TS and the possible
as being 68.89 kcal.mol . Through exception to the even-electron
2
m/z 145 forms (b, c and d) would justify the low intensity of the
m/z 101 signal in the MS/MS spectra. The fragment m/z 101
appeared at Elab higher than 30 eV, as observed in the works on
rule, as shown for Q2, the lapacholate anion should eliminate
the side chain as a radical, as observed in ESI investigations on
[
17]
Q4 (Scheme 10).
O
O
O
O
+
O
H
O
m/z 186
(
(
72.86)
68.89)
(59.07)
(72.42)
TS
E
O
O
O
O
O
O
m/z 243
m/z 243
(
0.0)
(1.51)
isolapacholate
(2.23)
lapacholate
-
Scheme 10. Potential energy surface for [Q3-H] fragmentation. Values are relative Gibbs energies. Italic values are relative enthalpies at 298 K. All the
ꢁ1
values are expressed in kcal.mol
.
J. Mass Spectrom. 2012, 47, 1648–1659
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

R. Vessecchi et al.
modeling of naphthoquinone derivatives with cytotoxic activity
in human promyelocytic leukemia HL-60 cell line. J. Med. Chem.
Conclusions
2
007, 50, 696.
The 2-hydroxy-1,4-naphthoquinone derivatives display interesting
dissociation pathways for their deprotonated and protonated ions.
The side chain plays an important role in the fragmentation of
these protonated and deprotonated compounds.
HBs influence the PA and GB values and may suggest the
different protonation sites and fragmentation pathways for each
analyzed compound. The AIM and NBO analyses contributed
to better understanding of the HB effect on the stability of
each conformer.
Our results are important for the description of lapachol
derivatives in biological matrices, biotransformation products
and new derivatives. All the data can be used during the MS
analysis of 2-hydroxy-1,4-naphthoquinone, to distinguish the
nature of the side chain at position C(3) of the quinonoid moiety.
The major fragmentation processes are crucial for the character-
ization of the side chain possibly present at position C(3). These
results, in combination with those described for lapachol and
other 1,4-naphthoquinones, can aid improvement of the knowl-
edge about this class of compounds, their reactivity in the gas
phase and analysis of their congeners, which can be found in
some plant species.
[
10] E. N. da Silva, M. A. B. F. de Moura, A. V. Pinto, M. D. F. R. Pinto, M. C. B. V.
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O. de Moraes, V. F. Ferreira, M. O. F. Goulart. Cytotoxic, trypanocidal
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C. Amatore. Electrochemical parameters and techniques in drug
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Chem. Commun. 2008, 23, 2612.
[12] T. Ossowski, P. Pipka, A. Liwo, D. Jeziorek. Electrochemical and UV-
spectrophotometric study of oxygen and superoxide anion radical
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Santos, H. P. Araújo, S. C. Bourguignon. Trypanocidal agents with
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Acknowledgements
Ricardo Vessecchi thanks FAPESP for the post-doctoral scholarship
2
009, 8, 1478.
(Grants 09/08281-3). Ricardo Vessecchi also thanks CAPES/PNPD.
17] R. Vessecchi, F. S. Emery, S. E. Galembeck, N. P. Lopes. Fragmentation
studies and electrospray ionization mass spectrometry of lapachol:
protonated, deprotonated and cationized species. Rapid Comm.
Mass Spectrum. 2010, 24, 2101.
18] M. N. Eberlin. Electrospray ionization mass spectrometry: a major tool
to investigate reaction mechanisms in both solution and gas-phase.
Eur. J. Mass Spectrom. 2007, 13, 19.
Authors thank the Brazilian foundations FAPESP, CAPES and CNPq
for financial support of this research.
Supporting information
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spectroscopy. Chem. Soc. Rev., 2012, 41, 547.
Supporting information may be found in the online version of
this article. All the geometries, energies and vibrational frequencies
obtained by means of the B3LYP/6-31 + G(d,p) model are available
as supporting information. All the spectral data (ESI-MS and ESI-MS/
MS) are also available as supporting information (Figs. S1–S13) and
from the correspondence author.
[
20] A. E. M. Crotti, E. S. Bronze-Uhle, P. G. B. D. Nascimento, P. M. Donate,
S. E. Galembeck, R. Vessecchi, N. P. Lopes. Gas-phase fragmentation
of gamma-lactone derivatives by electrospray ionization tandem
mass spectrometry. J. Mass Spectrom. 2009, 44, 1733.
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