A combined experimental and computational investigation on the unusual molecular mechanism of the lossen rearrangement reaction activated by carcinogenic halogenated quinones

Article abstract of DOI:10.1021/jo5022713

The classic Lossen rearrangement is a wellknown reaction describing the transformation of an Oactivated hydroxamic acid into the corresponding isocyanate. In this study, we found that chlorinated benzoquinones (C_nBQ) serve as a new class of agents for the activation of benzohydroxamic acid (BHA), leading to Lossen rearrangement. Compared to the classic one, this new kind of C_nBQ-activated Lossen rearrangement has the following unique characteristics: (1) The stability of C_nBQ-activated BHA intermediates was found to depend not only on the degree but also on the position of Cl-substitution on C_nBQs, which can be divided into two subgroups. (2) It is the relative energy of the anionic C_nBQ-BHA intermediates that determine the rate of this C_nBQ-activated rearrangement, which is the rate-limiting step, and the Cl or H ortho to the reaction site at C_nBQ is crucial for the stability of the anionic intermediates. (3) A pK_a-activation energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.

Full text of DOI:10.1021/jo5022713

Article
pubs.acs.org/joc
A Combined Experimental and Computational Investigation on the
Unusual Molecular Mechanism of the Lossen Rearrangement
Reaction Activated by Carcinogenic Halogenated Quinones
Guo-Qiang Shan,†,‡ Ao Yu,*,§ Chuan-Fang Zhao,§ Chun-Hua Huang,† Ling-Yan Zhu,‡
and Ben-Zhan Zhu*,†,¶
†State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, Beijing 100085, China
‡Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, Tianjin Key Laboratory of Environmental
Remediation and Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China
§Central Laboratory, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China
¶Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331, United States
S
* Supporting Information
ABSTRACT: The classic Lossen rearrangement is a well-
known reaction describing the transformation of an O-
activated hydroxamic acid into the corresponding isocyanate.
In this study, we found that chlorinated benzoquinones
(CnBQ) serve as a new class of agents for the activation of
benzohydroxamic acid (BHA), leading to Lossen rearrange-
ment. Compared to the classic one, this new kind of CnBQ-
activated Lossen rearrangement has the following unique
characteristics: (1) The stability of CnBQ-activated BHA
intermediates was found to depend not only on the degree but
also on the position of Cl-substitution on CnBQs, which can be divided into two subgroups. (2) It is the relative energy of the
anionic CnBQ−BHA intermediates that determine the rate of this CnBQ-activated rearrangement, which is the rate-limiting step,
and the Cl or H ortho to the reaction site at CnBQ is crucial for the stability of the anionic intermediates. (3) A pKa−activation
energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the
acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications
for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.
remained unclear. First, in that study, neither TCBQ O-
activated BHA intermediates (e.g., IN1 in Scheme 1) nor the
initial transient rearrangement product of BHA, phenyl
isocyanate (Ph-NCO), was directly isolated and identified
due to their extreme instability. Second, it was not clear
whether this kind of Lossen rearrangement reaction is a general
mechanism for enhancing dechlorination of all chlorinated
benzoquinones. Third, it was unclear what the major difference
of the halogenated quinone-activated Lossen rearrangement is
as compared to the classic one.
Therefore, in the present study, we tried to address the
following questions: (i) Is it possible to get the more stable O-
chloroquinonated BHA intermediates which are then unequiv-
ocally characterized when TCBQ is substituted with other less
chlorinated quinones, and if so, (ii) do these intermediates
decompose via the same Lossen rearrangement, and if so, under
INTRODUCTION
■
The Lossen rearrangement, which was first reported by Lossen
in 1872, is a thermal or alkaline conversion of hydroxamic acid
into isocyanate via the intermediacy of its O-activated (such as
O-acyl, -sulfonyl, or -phosphoryl) derivative.1−8 It has been
established that the O-activation of hydroxamic acids is
essential for Lossen rearrangement to take place.1−4 Recently,
we found that benzohydroxamic acid (BHA) was able to
dechlorinate tetrachloro-1,4-benzoquinone (TCBQ) via an
unusually mild and facile Lossen rearrangement mechanism
(Scheme 1).9 In that study, TCBQ and other tetrahalogenated
quinones were found to serve as a unique class of activating
agents for in situ activation of free hydroxamic acids. Compared
with the classic Lossen rearrangement reactions, this novel
variation of the Lossen rearrangement reaction took place at
room temperature and under neutral or even weakly acidic pH
and is responsible for the detoxification of the carcinogenic
tetrahalogenated quinones. However, some key issues of the
chloroquinone-activated Lossen rearrangement mechanism
Received: October 3, 2014
© XXXX American Chemical Society
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Scheme 1. Proposed Mechanism for the Dramatic Acceleration of TCBQ Hydrolysis by BHA: Suicidal Nucleophilic Attack
Coupled with an Unusual Double Lossen Rearrangement9
what experimental conditions? (iii) What is unique for the
halogenated quinone-mediated Lossen rearrangement as
compared to the classic one? (iv) Is there a correlation
between the pKa values of the corresponding conjugate acids of
the leaving groups, the hydroxylated choroquinones (Cn−1BQ-
OH, n = 1−4), and the stability of the O-chloroquinonated
BHA intermediates, and what is the underlying reason? (v)
What is the potential biological and environmental relevance of
this novel halogenated quinone-activated Lossen rearrange-
ment? To answer the above questions, in this study, a
combined experimental and theoretical investigation was
conducted to systematically examine the reactions of all seven
homologous series of chlorinated 1,4-benzoquinones (CnBQ, n
= 1−4) with BHA.
rearranged products, Cn−1BQ-OH, we would speculate that
when TCBQ is replaced with the less chlorinated benzoqui-
nones, for example, 2,5-dichloro-1,4-benzoquinone (2,5-
DCBQ), the rearrangement rate of 2,5-DCBQ O-activated
BHA derivatives should be much slower because of the weak
acidity of 2-chloro-5-hydroxy-1,4-benzoquinone (CBQ-OH,
pKa: 3.63, measured in this work) and 2,5-dihydroxy-1,4-
benzoquinone (pKa1: 2.95),12 which are the conjugate acids of
the leaving group for the reaction of 2,5-DCBQ/BHA, as
compared to TrCBQ-OH and DDBQ in TCBQ/BHA. If this is
the case, then we might further speculate that if the reaction
between 2,5-DCBQ and BHA took place in a way similar to
that for TCBQ/BHA, the 2,5-DCBQ O-activated BHA
intermediates of 2,5-DCBQ/BHA, the counterparts of IN1
and IN2 (Scheme 1), might be stable enough for direct
detection and identification.
Isolation and Identification of the Relatively Stable O-
Chloroquinonated BHA Derivatives of 2,5-DCBQ/BHA.
The reaction of 2,5-DCBQ with BHA was first studied by
quadrupole time-of-flight electrospray ionization mass spec-
trometry (Q-TOF-ESI-MS). We found that the major ion peak
for 2,5-DCBQ/BHA at a molar ratio of 1:1 is at m/z 157
(Figure S1A, Supporting Information (SI)), which was initially
assigned to the molecular peak of 2-chloro-5-hydroxy-1,4-
benzoquinone (CBQ-OH), the counterpart of TrCBQ-OH
(Scheme 1). Subsequent quantitative HPLC analysis, however,
revealed that the yield of CBQ-OH was only 2%, and the major
ion peak at m/z 157 might be the fragment ion of an unknown
product. Special attention was then paid to the weak ion peak at
m/z 276, which was neglected at first due to its low abundance
(only 5% of the major ion peak) (Figure S1A, SI). Another
weak ion peak at m/z 377 was also observed in the MS spectra
of 2,5-DCBQ/BHA (1:2) (Figure S1B, SI). On the basis of
RESULTS AND DISCUSSION
■
Just as described in our previous study,9 the proposed TCBQ
O-activated BHA intermediate, IN1, was unable to be isolated
and identified. The instability is possibly due to the unusually
rapid and facile decomposition via Lossen rearrangement to
2,3,5-trichloro-6-hydroxy-1,4-benzoquinone (TrCBQ-OH) and
Ph-NCO. For the classic Lossen rearrangement reactions
activated by the acyl, sulfonyl, or phosphoryl group, it has been
found that the rearrangement rate is directly proportional to the
acidity of the conjugate acid of the leaving group.1−4 Due to the
strong acidity of TrCBQ-OH (pKa: 1.0910) and 2,5-dichloro-
3,6-dihydroxy-1,4-benzoquinone (DDBQ, pKa1: 0.5811 or
0.7612), which are the conjugate acids of the leaving anions
in that study, it was expected that the rearrangement of the
postulated reaction intermediates should be very fast so that the
intermediates rearrange rapidly once formed.
If there indeed exists such a correlation between the
rearrangement rate and the acidity of the corresponding
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molecular mass calculations, the weak ion peaks at m/z 276 and
m/z 377 should actually correspond to the single- (P1 in
Scheme 2) and double-substituted (P2 in Scheme 2) adducts of
2,5-DCBQ with BHA, respectively.
Scheme 2. Proposed Mechanism for 2,5-DCBQ/BHA
Reaction
Figure 1. Isolation and identification of the relatively stable O-
chloroquinonated BHA derivatives of 2,5-DCBQ/BHA. HPLC
chromatograms of 2,5-DCBQ/BHA (1:1 or 1:2) in CH3COONH4
buffer (pH 7.4, 0.1 M) at 275 nm (A); MS spectrum of P1 at the
retention time of 6.53 min in the HPLC chromatogram (B); MS
spectrum of P2 at the retention time of 9.00 min in the HPLC
chromatogram (C).
Lossen rearrangement reaction. The experimental activation
energy of the rearrangement of P1 was calculated to be 23.46
kcal/mol, according to the measured initial kinetics at 25/30/
35/40 °C and the Arrhenius equation. Further, we found that
decomposition of P1 in aqueous buffer is just through the same
Lossen rearrangement mechanism, because the analysis by TLC
and HPLC (Figure 2D) showed that the major decomposition
products are aniline, N,N′-diphenylurea, and CBQ-OH, which
are typical products of Lossen rearrangement reactions.
To test whether this assignment is the case, the reaction of
2,5-DCBQ/BHA was then investigated in detail by HPLC/Q-
TOF-ESI-MS. It was found that the addition of 2,5-DCBQ to
BHA at different molar ratios indeed rapidly led to the
formation of the two final products P1 and P2. The major
reaction product for 2,5-DCBQ/BHA at a 1:1 ratio is P1 with
the retention time of 6.53 min (Figure 1A), which shows the
molecular ion [M − H]− at m/z 276 and the fragment ion at
m/z 157; both of them are one-chlorine-isotope peak clusters
(Figure 1B). The major product for 2,5-DCBQ/BHA at ≤1:2
ratios is P2 with the retention time of 9.00 min (Figure 1A),
which has the molecular ion [M − H]− at m/z 377 and the
fragment ions at m/z 258 and m/z 139 (Figure 1C). Although
the collision energy was lowered to 3.0 V, the abundance of
molecular ion peak of P1 or P2 was still much lower than their
respective fragment ion peaks, indicating that the two products
were readily fragmented even under very mild MS conditions.
Unfortunately, we still failed to directly detect and identify
the transient Ph-NCO due to its extreme instability in aqueous
buffer. So, to detect this initial Lossen rearrangement product,
the key evidence for Lossen rearrangement, we performed the
decomposition of P1 under nonaqueous conditions (for details
on how to detect Ph-NCO via pyrolysis of P1, see Supporting
Information). As expected, Ph-NCO was detected by GC/MS
with a retention time at 6.75 min (Figure 2E) and a
characteristic MS spectra with peaks at m/z 119 (100%), 91
(41%), and 64 (24%), the same as that for authentic Ph-NCO.
Proposed Molecular Mechanism for the Reaction of
2,5-DCBQ/BHA and Comparison with That of TCBQ/
BHA. On the basis of the above experimental results, the
reaction pathways for 2,5-DCBQ/BHA in aqueous solution was
proposed as the following (Scheme 2): a nucleophilic reaction
takes place between 2,5-DCBQ and the benzohydroxamate
anion (BHA−) (at high 2,5-DCBQ/BHA molar ratios), forming
the relatively stable single-substituted P1 (pKa 5.0, measured in
this work) at room temperature and under neutral pH.
Following loss of a proton from nitrogen to form the anionic
intermediate of P1, a slow decomposition undergoes Lossen
rearrangement to form CBQ-OH and Ph-NCO. The rearrange-
ment rate is markedly enhanced under alkaline pH or at higher
temperatures. Once formed, the rearranged product Ph-NCO
rapidly hydrolyzes to form aniline, which then reacts with
another Ph-NCO to yield N,N′-diphenylurea. When BHA is in
excess, a second nucleophilic reaction between P1 and BHA
occurs, forming the double-substituted P2.
1
P1 or P2 was further identified by H and 13C NMR as the
single- and double-substituted 2,5-DCBQ adducts with BHA,
respectively (Figure S2, Figure S3, and Table S1 in Supporting
Information).
Decomposition of P1 via Lossen Rearrangement at
Higher Temperature or Alkaline pH. An interesting
question to investigate is whether the stable 2,5-DCBQ O-
activated BHA derivative P1 would decompose through the
same Lossen rearrangement. We found that aqueous P1
decomposed with a half-life of approximately 2.5 h at room
temperature in neutral buffer (pH 7.0) (Figure 2A), which is in
contrast to the extremely rapid decomposition of IN1 in the
TCBQ/BHA reaction. Interestingly, the slow decomposition of
P1 was markedly accelerated by higher temperature or alkaline
pH (Figure 2B and 2C), which is consistent with the classic
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Figure 2. Decomposition of P1 via Lossen rearrangement at higher temperature or alkaline pH. The formation of CBQ-OH was accompanied by the
relatively slow decomposition of P1 in neutral solution (A, pH 7.0) at 25 °C. The decomposition of P1 in aqueous buffer was markedly accelerated
(B) at higher temperature and (C) under alkaline conditions. (D) HPLC chromatogram of the thermolysis of P1 in buffer solution (pH 8.0) at 60
°C, compared to that of N,N′-diphenylurea and CBQ-OH as references. (E) The GC/MS chromatogram of pyrolysis product phenyl isocyanate of
P1, compared to that of authentic phenyl isocyanate.
Comparative analysis of the reaction mechanisms between
TCBQ/BHA and 2,5-DCBQ/BHA indicated that the stability
of O-chloroquinonated BHA derivatives seems to determine
the reaction pathway. At high molar ratios (≥1), the reaction
pathway of 2,5-DCBQ/BHA is the same as that of TCBQ/
BHA, entailing a nucleophilic attack and then a Lossen
rearrangement. The only difference is that the rearrangement
rate of the relatively stable derivative P1 is much slower than
that of the transient intermediate IN1 in TCBQ/BHA.
Therefore, when BHA is in excess, P1 is trapped by excessive
BHA− to give the double-substituted product P2, while IN1
rearranges rapidly into TrCBQ-O− (8, Table S2, SI) and Ph-
NCO. Then TrCBQ-O− reacts with excessive BHA− via the
second nucleophilic attack coupled with the second Lossen
rearrangement to give the final product DDBQ (9, Table S2,
SI).
Isolation and Identification of Other O-Chloroquino-
nated BHA Derivatives. Similar relatively stable 1:1 adducts
with BHA were isolated and identified, when 2,5-DCBQ was
replaced by its isomer 2,6-DCBQ, which showed the molecular
ion peak [M − H]− at m/z 276, and the fragment ion at m/z
157, both of which were one-chlorine-isotope peak clusters
(Figure S4A and S4B, SI), but not with another isomer 2,3-
DCBQ. Instead, the rearranged product 2-chloro-3-hydroxy-
1,4-benzoquinone (2, Table S2, SI) was isolated from 2,3-
DCBQ/BHA, which showed the molecular ion peak [M − H]−
at m/z 157 (Figure S4A and S4D, SI). Thus, it would be
interesting to know why these O-activated BHA derivatives of
these DCBQ isomers have different stabilities.
isomers (2,5-, 2,6-, and 2,3-DCBQ) (in 1:1 ratio) should be 2-
chloro-5-hydroxy- (3, Table S2, SI), 2-chloro-6-hydroxy- (4,
Table S2, SI), and 2-chloro-3-hydroxy-1,4-benzoquinone (2,
Table S2, SI); their pKa values are found to be 3.63 (expt), 3.65
(calcd), and 2.28 (expt), respectively (Table S2, SI). From
these data, we speculated that when the pKa value is ≤2.5, then
the O-chloroquinonated BHA derivatives might be unstable
and may quickly decompose through Lossen rearrangement to
form the corresponding Cn−1BQ-OH and Ph-NCO, but when
the pKa value is >2.5, then the O-chloroquinonated BHA
derivatives might be stable enough to be isolated and identified.
To test the above hypothesis, we studied the reactions
between BHA and two other chlorinated benzoquinones: One
is the less chlorinated 2-chloro-1,4-benzoquinone (2-CBQ),
and the other is the more chlorinated 2,3,5-trichloro-1,4-
benzoquinone (TrCBQ). If the above hypothesis were true,
then we would expect that 2-CBQ should be able to form
relatively more stable 1:1 adduct with BHA because the pKa of
its corresponding 2-hydroxy-1,4-benzoquinone (1, Table S2,
SI) is 4.0−4.2.13−15 For TrCBQ, if substituted at the 5-chloro
position by BHA, we would expect it should also be able to
form a relatively stable 1:1 adduct with BHA because the pKa of
its corresponding 2,3-dichloro-5-hydroxy-1,4-benzoquinone (5,
Table S2, SI) is 2.89, but if substituted at the 2- or 3-chloro
position, then no stable 1:1 TrCBQ−BHA adducts would be
isolated because the pKa values of the corresponding Cn−1BQ-
OH are 1.88 (2,5-dichloro-3-hydroxy-1,4-benzoquinone, 6,
Table S2, SI) and 1.5716 (2,6-dichloro-3-hydroxy-1,4-benzo-
quinone, 7, Table S2, SI), respectively. We found that this was
indeed the case (see Figure S5 in Supporting Information for
details on how to isolate and characterize the products of other
CnBQ/BHA).
As mentioned above, it was reported that there might be a
correlation between the rate of Lossen rearrangement and the
acidity of the conjugate acid of the leaving group.1−4 In the
current work, the corresponding conjugate acids of the leaving
group for the O-activated BHA adducts with three DCBQ
Therefore, we can expect that the stability of the O-
chloroquinonated BHA derivatives should follow the general
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rule that the more acidic the conjugate acids of the leaving
groups, the faster the rearrangement rates. This also suggests
that although the reactions of CnBQ/BHA (1:1) follow the
same pathway, the rearrangement rate of the O-chloroquino-
nated BHA derivatives are very different. On the basis of the
difference between the stability of the O-chloroquinonated
BHA derivatives, CnBQs can be classified as two subgroups
(Figure 3): TCBQ-type and DCBQ-type. The former,
ing step but can be overcome easily under room temperature.
This is consistent with the experimental results that TCBQ
reacts with BHA completely at room temperature to form the
rearranged products within 1 min.9
The calculation data of the reaction of 2,5-DCBQ/BHA are
also very consistent with the experimental results (Figure 4B).
When the reaction molar ratio of 2,5-DCBQ/BHA is 1:1, the
activation and deprotonation steps are facile with an activation
energy of 10.14 and 9.43 kcal/mol, respectively. The
subsequent Lossen rearrangement ((D)-IN2 (D, short for
DCBQ) to CBQ-O−) is also the rate-determining step with a
calculated activation energy of 23.90 kcal/mol (Figure 4B1),
which is in complete agreement with the experimental
activation energy of 23.46 kcal/mol and is 6.93 kcal/mol
higher than that of the first Lossen rearrangement of TCBQ/
BHA. Therefore, compared with (T)-IN2, the anionic 2,5-
DCBQ O-activated BHA intermediates (D)-IN2 should not
quickly decompose via Lossen rearrangement and can be
isolated as substituted adduct P1.
When BHA is in excess, accompanied by the slow
rearrangement of (D)-IN2, the second nucleophilic attack of
BHA− to form double-substituted product P2 occurs (Figure
4B2), which is facile with an activation energy of 15.66 kcal/
mol, 8.24 kcal/mol lower than that of the concurrent Lossen
rearrangement step ((D)-IN2 to CBQ-O−). This can explain
why the 2,5-DCBQ O-activated BHA intermediates did not
prefer Lossen rearrangement (but rather nucleophilic sub-
stitution) as (T)-IN2 when BHA is in excess. This is also in
good agreement with the experimental results: Quantitative
determination by HPLC using the purified authentic P1 and P2
as standard reference showed that the reaction of 2,5-DCBQ/
BHA (1:1) led to the formation of 83% P1 and 6% P2 within 1
min, while the reaction of 2,5-DCBQ/BHA (1:2) yielded 78%
P2 and 16% P1.
The DFT calculations for other CnBQ/BHA reactions were
also performed. The calculation results of the first nucleophilic
reaction coupled with Lossen rearrangement when the molar
ratio of CnBQ/BHA is 1:1 are summarized in Figure 5 and
Table S3 (SI). The reaction of CnBQ/BHA (1:1) entailed a
facile nucleophilic attack (CnBQ to IN1 via TS1) with the
activation energy of 5−13 kcal/mol and facile deprotonation of
N−H to form its corresponding anionic CnBQ-activated BHA
intermediate IN2 and a subsequent rate-determining Lossen
rearrangement (IN2 to Cn−1BQ-OH via TS2) with the
activation energy of 16−25 kcal/mol.
Careful examination of the potential energy profiles of the
Lossen rearrangement pathway of CnBQ/BHA (1:1) in Figure
5 reveals that the discrepancy of activation energies in the
rearrangement step (IN2 to TS2) is mainly due to the relative
energies of anionic intermediates IN2 (ΔΔG > 6 kcal/mol),
rather than the relative energies of the transition state TS2
(ΔΔG < 1 kcal/mol). Interestingly, it was observed that the
relative energies of IN2 fall into two subgroups: IN2 of 2,3-
DCBQ, TrCBQ-2, TrCBQ-3, and TCBQ have relative energies
lower than that of IN2 of CBQ, 2,5-DCBQ, 2,6-DCBQ, and
TrCBQ-5, which is in agreement with our above experimental
classification (TCBQ-type and DCBQ-type in Figure 3). From
the structures of these two groups of CnBQ, a specific
distinction is observed: TCBQ-type have an o-chlorine adjacent
to the reaction site while DCBQ-type CnBQs have an o-
hydrogen.
Figure 3. CnBQs are classified by the stability of the CnBQ-activated
intermediates with BHA (1:1, * reaction site).
containing 2,3-DCBQ, TrCBQ-2, TrCBQ-3, and TCBQ, reacts
with BHA just as for TCBQ/BHA to form the transient CnBQ/
BHA (1:1) intermediate, which is unable to be isolated and
identified by LC/MS due to its rapid decomposition via
rearrangement, while the corresponding CnBQ/BHA (1:1)
derivatives of the latter, containing CBQ, 2,5-DCBQ, 2,6-
DCBQ, and TrCBQ-5, are stable enough to be isolated and
identified under our experimental conditions because of the
slow decomposition rate just as for 2,5-DCBQ/BHA.
DFT Study of the Reaction Mechanism of CnBQ/BHA.
Although we performed an extensive experimental study on the
reactions of CnBQ/BHA, some questions raised were not yet
solved satisfactorily: (i) What is unique for the halogenated
quinone-mediated Lossen rearrangement as compared to the
classic one? (ii) Why is the stability of the O-chloroquinonated
BHA derivatives so different? (iii) Why is there a correlation
between the rearrangement rates of the O-chloroquinonated
BHA derivatives and the pKa of the rearranged products
C
n−1BQ-OHs?
To pursue answers to these questions, we performed a
theoretical investigation on the intermediates and energies of
the reactions of CnBQ/BHA (1:1). The calculation of the
reaction of TCBQ/BHA (Figure 4A) reproduces the
experimental results very well. The first step is the nucleophilic
attack of BHA− on TCBQ via the transition state (T)-TS1 (T,
short for TCBQ) forming the neutral intermediate (T)-IN1
with an energy barrier of 5.79 kcal/mol, and then the second
step is deprotonation of N−H of (T)-IN1 forming the anionic
intermediate (T)-IN2 with an energy barrier of 4.17 kcal/mol.
Subsequent conversion of (T)-IN2 into TrCBQ-O− (via (T)-
TS2) requires an activation energy of 16.97 kcal/mol. When
BHA is in excess, TrCBQ-O− further reacts with BHA through
the second Lossen rearrangement, yielding DDBQ and another
molecule of Ph-NCO. From the potential energy surface of the
overall reaction pathway, it is easy to find that the first Lossen
rearrangement ((T)-IN2 to TrCBQ-O−) is the rate-determin-
It has been shown that the rate of Lossen rearrangement of
hydroxamic acids is related to the electron-withdrawing
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Figure 4. DFT study of the reaction mechanism of CnBQ/BHA. The potential energy surface of the reactions of TCBQ/BHA (A); 2,5-DCBQ/BHA
(1:1, B1) and (1:2, B2).
substituents in R′ of diacyl hydroxylamines (R-C(O)NH-O-
R′) when R and R′ are aryl groups.17−19 Compared with that of
the rearrangement activation energy of R-C(O)NH-O-R′,
which showed that when R′ is o-NO2 (or Br, Cl)-C6H4, the
activation energy is about 1 kcal/mol less than that when R′ is
C6H5 and o-CH3OC6H4;18 however, the discrepancy between
the activation energy of DCBQ-type IN2 and TCBQ-type IN2 is
relatively large (about 5 kcal/mol). This suggests that the
electron-withdrawing effect of the o-chlorine substituent in the
quinoid ring might not be the sole feasible factor to influence
the rearrangement rate. Therefore, we re-examined in-depth the
structures of (T)-IN2 and (D)-IN2 (Figure 6).
From the structure of (T)-IN2 (Figure 6A), it was observed
that the electrostatic repulsion between the anionic nitrogen
and the neighboring chlorine atom made the benzamide twist
out 20° from the quinone plane, and the chlorine atom moved
about 7.7° from the quinone plane in the opposite direction.
The twisted structure of the anionic intermediate (T)-IN2 is
unstable, because it breaks the resonance of the quinone and
benzamide parts; therefore, it easily undergoes rearrangement.
However, (D)-IN2 is a planar molecule (Cs symmetry, Figure
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Figure 5. Potential energy (ΔG) surface of the reactions of CnBQ/BHA (1:1).
Figure 7. pKa−activation energy correlation between the pKa of
Cn−1BQ-OH and the activation energy for the Lossen rearrangement
of CnBQ/BHA. For the numbering of the hydroxylated benzoquiones,
see Table S2, SI. The experimental pKa values were preferred for linear
fitting.
energy correlation indicates that the pKa of the corresponding
n−1BQ-OH might also be taken into account in consideration
of the “ortho effect”. For DCBQ-type Cn−1BQ-OH, the acidity
is only affected by the electron-withdrawing effect of m- or p-
chlorine. However, the acidity of TCBQ-type Cn−1BQ-OH is
mainly influenced by o-chlorine. It seems reasonable that the o-
chlorine effect might be much stronger than m- or p-chlorine.
Summary. In this study, through a combined experimental
and theoretical investigation, we found that all seven isomers of
chlorinated benzoquinones (CnBQs) can serve as a new class of
agents for the activation of free hydroxamic acids, leading to
Lossen rearrangement. Compared to the classic one, this newly
discovered CnBQ-activated Lossen rearrangement has the
following three unique characteristics: (1) The stability of
CnBQ-activated BHA intermediates was found to (i) be
dependent not only on the degree but also on the position of
chloro-substitution on the quinone structure of CnBQ, which
can be divided into two subgroups: TCBQ- and DCBQ-type,
and to (ii) follow the general rule in the correlation between
the rearrangement rates and the acidity of the rearranged
products, the hydroxlated benzoquinones, whose pKa values
vary remarkably from 0.6 to 4.2. (2) The deprotonation of N−
H to form its anionic CnBQ-activated BHA intermediate is
necessary for successive rearrangement. Interestingly and
unexpectedly, we found that it is the relative energy of the
anionic intermediates that determine the rate of this CnBQ-
activated Lossen rearrangement, which is the rate-limiting step
(while for classic Lossen rearrangement, the rate-limiting step
C
Figure 6. Structures of IN2 of TCBQ-type (A) and of DCBQ-type
(B).
6B) with no electrostatic repulsion, so the conjugation
interaction stabilizes the anion intermediate. This kind of
stereoelectronic effect is also present in IN2 of all other TCBQ-
type or DCBQ-type CnBQ (Figure 6). Thus, this suggests that
chlorine or hydrogen at a position ortho to the reaction site
might be the pivotal factor to determine the relative energy and
then the rearrangement rate, which is the unique feature of
these halogenated quinone-mediated Lossen rearrangements.
Correlation between the Rate of Lossen Rearrange-
ment and the Acidity of Cn−1BQ-OH. It is obvious that the
rate of rearrangement was determined by the stability of the
anionic CnBQ O-activated BHA intermediate. Then, why is
there the relationship between the Lossen rearrangement rate
and the pKa of Cn−1BQ-OH? To answer this question, we
calculated the pKa of all Cn−1BQ-OHs (Table S2, SI), and
interestingly, we found that the pKa of Cn−1BQ-OH has a good
linear relationship with the activation energy for the Lossen
rearrangement of CnBQ/BHA (Figure 7). The pKa−activation
G
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has been generally considered to be the activation of the
hydroxamic acid by various activating agents), and the chlorine
or hydrogen ortho to the reaction site at CnBQ is crucial for the
stability of the anionic intermediates. (3) There exists a pKa−
activation energy correlation for this CnBQ-activated Lossen
rearrangement reaction, which can explain why the correlation
exists between the rate of the rearrangement and the acidity of
the conjugate acid of the anionic leaving group.
Potential Biological and Environmental Implications.
Halogenated quinones represent a class of toxicological
intermediates that can create a variety of hazardous effects in
vivo, including acute hepatoxicity, nephrotoxicity, and carcino-
genesis.20−22 Chlorinated benzoquinones (CnBQs) are the
major genotoxic and carcinogenic quinoid metabolites of the
widely used pesticides chlorophenols such as the wood
preservative pentachlorophenol (PCP) and 2,4,5-trichlorophe-
nol. CnBQs have also been observed as reactive oxidation
intermediates or products in processes used to oxidize or
destroy chlorophenols and other polychlorinated persistent
organic pollutants (POPs) in various chemical and enzymatic
systems.20−22 Recently, several CBQs were identified as new
chlorination disinfection byproducts in drinking water and in
swimming pool waters.23,24
Hydroxamic acids have attracted considerable interest
recently because of their capacity to inhibit a variety of
enzymes, such as metalloproteases and lipoxygenase, and
transition metal-mediated oxidative stress.4,9,25,26 Many of the
activities of these hydroxamic acids are thought to be due to
their metal-chelating properties. In addition to metal chelation,
hydroxamic acids are considered to be good α-nucleophiles.
We have shown previously that hydroxyl (or alkoxyl) and
carbon-centered quinone ketoxy radicals (leading to DNA
damage) and chemiluminescence can be produced during the
metal-independent decomposition of H2O2 (or organic hydro-
peroxides) by TCBQ and other halogenated quinoid
carcinogens.27−32 Recently, we found that the formation of
these reactive free radicals and TCBQ-induced cellular toxicity
were markedly inhibited by benzohydroxamic acid (BHA) and
other hydroxamic acids,33,34 via the unusually facile two-
consecutive-step Lossen rearrangement mechanism.9 It has
been well documented that such radical damage processes
(radical oxidations) occur as autocatalyzed chain reactions.35
Whereas, most often, the focus of radical suppression is by
inhibiting radical propagation,36 the presented strategy relies on
inhibiting radical initiation reactions, i.e., the halogenated
quinone-supported homolytical cleavage of peroxides. This is
conceptually similar to the iron-chelating efforts for prevention
of food spoilage.37
As demonstrated in the present and previous study,
hydroxamic acids, in addition to BHA, might be especially
suited for detoxification of halogenated quinone carcinogens via
the Lossen rearrangement mechanism. Of particular interest in
this regard is the fact that two hydroxamic acids are already
approved for clinical applications, deferoxamine for iron
overload and suberoylanilide hydroxyamic acid (Vorinostat),
recently approved for cutaneous T-cell lymphoma.4,9,25,26 Thus,
further investigation is needed to determine whether
hydroxamic acids can be used safely and effectively as
prophylactics for the prevention or treatment of human
diseases such as liver and bladder cancer associated with the
toxicity of polyhalogenated quinoid carcinogens.
EXPERIMENTAL AND COMPUTATIONAL
METHODS
■
Chemicals. 2,5-Dichloro-1,4-benzoquinone (2,5-DCBQ), 2,6-di-
chloro-1,4-benzoquinone (2,6-DCBQ), 2-chloro-1,4-benzoquinone
(2-CBQ), tetrachloro-1,4-benzoquinone (TCBQ), benzohydroxamic
aicd (BHA), phenyl isocycanate (Ph-NCO), N,N′-diphenylurea, and
aniline were used as purchased. 2-Chloro-5-hydroxy-1,4-benzoquinone
(CBQ-OH), 2,3-dichloro-1,4-benzoquinone (2,3-DCBQ), and 2,3,5-
trichloro-1,4-benzoquinone (TrCBQ) were synthesized by our
research group according to the literature methods.38,39
Analysis of the Reaction of 2,5-DCBQ/BHA. The reaction
products of 2,5-DCBQ/BHA were analyzed with high-performance
liquid chromatography combined with electrospray ionization quadru-
pole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS). The
HPLC system was equipped with a photodiode array detector. For
direct MS analysis, a small portion (20 μL) of reaction solution of 1
mM 2,5-DCBQ with 1, 2, or 4 mM BHA in 1 mL of Chelex-treated
CH3COONH4 buffer (100 mM, pH 7.0) at room temperature during
the reaction period of 0−30 min was injected into the mass
spectrometer. All other MS experimental parameters were the same
as described previously.9 The yield of 2-chloro-5-hydroxy-1,4-
benzoquinone (CBQ-OH) from 2,5-DCBQ/BHA was quantified by
HPLC using synthesized CBQ-OH as standard according to the
previous method.28 For HPLC/MS analysis, the reaction solution was
injected into an LC-18 C18 column (5 μm, 4.6 × 250 mm) eluted by
the mobile phase (50 mM aqueous acetic acid and acetonitrile at
50:50) at a rate of 1.0 mL/min, and the chromatographic eluant was
monitored at 200−600 nm and then led to the mass spectrometer
through a splitter.
Isolation of the Major Reaction Products (P1 and P2) of 2,5-
DCBQ/BHA and the Identification of Decomposition Products
of P1 in Aqueous Solution. P1 and P2 were isolated by both
semipreparative HPLC and column chromatography. Milligram-scale
collection of P1 and P2 (Scheme 2) was performed with semi-
preparative HPLC apparatus equipped with a UV detector. The
reaction solution of 2,5-DCBQ/BHA (1:1 or 1:2, 1 mM 2,5-DCBQ)
in 1 mL of Chelex-treated CH3COONH4 buffer (100 mM, pH 7.0) at
room temperature after a reaction time of 5 min was injected into a
Prep-C18 semipreparative HPLC column (15 cm × 10.0 mm, 3 μm).
The mobile phase was 50 mM aqueous acetic acid−acetonitrile
(50:50) at a flow rate of 3.0 mL/min. The fractions were monitored at
275 nm and collected manually. Then collected fractions were
evaporated to eliminate acetonitrile and then extracted with ethyl
acetate. The collected ethyl acetate layer was dried over anhydrous
MgSO4 and evaporated to dryness under vacuum. Gram-scale P1 and
P2 were isolated by column chromatography. A solution of 2,5-DCBQ
(5 mM, 0.885 g) in acetonitrile (10 mL) was added dropwise to 100
mL of Chelex-treated CH3COONH4 buffer (100 mM, pH 7.0)
containing BHA (5 mM, 0.685 g) at room temperature. After the
mixture was stirred for 5 min, the solid was separated by filtration and
purified by silica gel column chromatography with tetrahydrofuran/
petroleum ether (1:9) as eluent. Preparation of P2 was carried out as
for P1 except that the molar ratio of 2,5-DCBQ/BHA was 1:2 and the
purification was carried out by recrystallization from tetrahydrofuran/
petroleum ether. Product P1 was golden-yellow and P2 was purple-red,
1
and their purity was 98% as determined using HPLC. H NMR and
13C NMR spectra of P1 and P2 were recorded at 400 and 101 MHz,
respectively, using tetramethylsilane ((CH3)4Si) as internal standard
and DMSO-d6 as solvent. Product P1: 1H NMR δ = 6.50 (s, 1H), 7.36
(s, 1H), 7.54 (m, 2H), 7.64 (m, 1H), 7.88 (m, 2H), 12.85 (s, 1H); 13C
NMR δ = 127.7, 128.8, 130.6, 132.6, 143.5, 158.3, 165.8, 178.7, 179.4.
1
Product P2: H NMR δ = 6.32 (s, 2H), 7.55 (m, 4H), 7.64 (m, 2H),
7.89 (m, 4H), 12.85 (s, 2H); 13C NMR δ = 127.7, 128.8, 130.5, 132.6.
158.4, 165.5, 180.5. For details, see Supporting Information.
P1 (1 mM) in PB buffer (0.1 mM, pH 8.0) was heated in 60 °C
water bath for 2 min and then spotted on analytical thin-layer
chromatography (TLC) plates or injected into an HPLC instrument.
TLC was carried out on silica gel plates with F-254 indicator.
Reactions were monitored by TLC using ethyl acetate−petroleum
H
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Article
ether (2:1) as the developing solvent with P1, and reagent-grade N,N′-
diphenylurea and aniline as standard references. The product spots on
TLC keeping pace with N,N′-diphenylurea or aniline were scraped and
extracted with ether for MS analysis. MS showed that N,N′-
diphenylurea ([M + H]+ at m/z 213) and aniline ([M + H]+ at m/
z 94) were formed during the thermolysis. P1’s thermolysis products
were also detected using HPLC with a mobile phase of 50 mM
aqueous acetic acid−acetonitrile at 60:40, and the chromatographic
eluant was monitored at 275 nm. Sample retention times were
compared to those of 2-chloro-5-hydroxy-1,4-benzoquinone (CBQ-
OH) and N,N′-diphenylurea as standard references. The thermolysis
kinetics of P1 was quantified based on HPLC by the external
standardization with isolated P1 and synthesized CBQ-OH.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: bzhu@rcees.ac.cn.
*E-mail: esr@nankai.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work in this paper was supported by the Strategic Priority
Research Program of CAS Grant No. XDB01020300, NSF
China Grants (21237005, 21321004, 20925724, 21077058;
21477139, 21407163), Tianjin Municipal Science and Tech-
nology Commission (12JCYBJC16000, 14JCZDJC40300), The
Foundation of State Key Laboratory of Environmental
Chemistry and Ecotoxicology (KF2012-15), Postdoctoral
Science Foundation of China (2014M561078), and NIH
Grants (ES11497, RR01008, and ES00210) (B.Z.).
Computational Methods. All of the computations were
performed using Gaussian 09.40 Geometry optimization and
corresponding harmonic vibration frequency calculations were
executed without any constraints using the B3LYP method41−44 with
6-31+G(d,p) basis set45−48 in the gas phase. All transition states were
characterized by one imaginary vibration frequency and the
intermediates with no imaginary frequency. Intrinsic reaction
coordinate (IRC) calculations were performed on the transition
state structures to confirm that the transition state was connected to
the correct reactant and product along the reaction paths. Solvent
effects were included by performing single-point energy calculations
(Esol) on the gas-phase optimized geometries with the CPCM49−52
model and UAKS radii in water at M06-2X/6-31+G(d,p) level of
theory. Truhlar’s M06-2X53,54 functional was developed for
computations involving main-group thermochemistry, kinetics, and
noncovalent interactions, which are important in this work. All of the
energies discussed in this paper and the Supporting Information are
relative Gibbs free energies (ΔGsol) in water solution at 298 K. The
relative enthalpy (ΔHsol) values in solution are also provided for
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HA + H2O = H3O+ + A
−
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(1)
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RT ln(10)
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ASSOCIATED CONTENT
■
S
* Supporting Information
The details on analysis of the reactions of other chlorinated
benzoquinones with BHA, the experimental measurement of
pKa values of P1 and some hydroxylated chloroquinoid
products, the NMR data of P1 and P2, and GC/MS detection
of phenyl isocyanate from P1 pyrolysis. This material is available
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