Chlorination increases the persistence of semiquinone free radicals derived from polychlorinated biphenyl hydroquinones and quinones

Article abstract of DOI:10.1021/jo801397g

(Chemical Equation Presented) Polychlorinated biphenyls (PCBs) comprise a group of persistent organic pollutants that differ significantly in their physicochemical properties, their persistence, and their biological activities. They can be metabolized via

Full text of DOI:10.1021/jo801397g

Chlorination Increases the Persistence of Semiquinone Free Radicals
Derived from Polychlorinated Biphenyl Hydroquinones and
Quinones
Yang Song,†,‡ Garry R. Buettner,‡,§ Sean Parkin,| Brett A. Wagner,‡ Larry W. Robertson,†,§
and Hans-Joachim Lehmler*,†,§
Department of Occupational and EnVironmental Health, The UniVersity of Iowa, 100 Oakdale Campus,
124 IREH, Iowa City, Iowa 52242-5000, Free Radical and Radiation Biology Program & ESR Facility,
The UniVersity of Iowa, Iowa City, Iowa 52242-1101, Interdisciplinary Graduate Program in Human
Toxicology, The UniVersity of Iowa, Iowa City, Iowa 52242-5000, and Department of Chemistry,
UniVersity of Kentucky, Lexington, Kentucky 40506-0055
hans-joachim-lehmler@uiowa.edu
ReceiVed July 1, 2008
Polychlorinated biphenyls (PCBs) comprise a group of persistent organic pollutants that differ significantly
in their physicochemical properties, their persistence, and their biological activities. They can be
metabolized via hydroxylated and dihydroxylated metabolites to PCB quinone intermediates. We have
recently demonstrated that both dihydroxy PCBs and PCB quinones can form semiquinone radicals (SQ•-)
in vitro. These semiquinone radicals are reactive intermediates that have been implicated in the toxicity
of lower chlorinated PCB congeners. Here we describe the synthesis of selected PCB metabolites with
differing degrees of chlorination on the oxygenated phenyl ring, e.g., 4,4′-dichloro-biphenyl-2,5-diol,
3,6,4′-trichloro-biphenyl-2,5-diol, 3,4,6,-trichloro-biphenyl-2,5-diol, and their corresponding quinones.
In addition, two chlorinated o-hydroquinones were prepared, 6-chloro-biphenyl-3,4-diol and 6,4′-dichloro-
biphenyl-3,4-diol. These PCB (hydro-)quinones readily react with oxygen or via comproportionation to
yield the corresponding semiquinone free radicals, as detected by electron paramagnetic resonance
spectroscopy (EPR alias ESR). The greater the number of chlorines on the (hydro-)quinone (oxygenated)
ring, the higher the steady-state level of the resulting semiquinone radical at near neutral pH.
Introduction
scale by batch chlorination of biphenyl in the presence of an
iron catalyst. They have been (and still are) used as coolants
and insulating fluids for transformers and capacitors. PCBs have
also been sold commercially for use as stabilizing additives in
flexible PVC coatings of electrical wiring and electronic
components, cutting oils, flame retardants, hydraulic fluids,
sealants, and adhesives. These industrial applications exploit
the physicochemical properties of PCBs, such as the high
thermal and chemical stability and high dielectric constant. The
commercial production and sale of PCBs were banned in the
United States in the late 1970s because of their persistence and
Polychlorinated biphenyls (PCBs) are a class of organic
compounds containing one to ten chlorine substituents covalently
attached to biphenyl.1,2 PCBs have been produced in industrial
† Department of Occupational and Environmental Health, The University of
Iowa.
‡ Free Radical and Radiation Biology Program & ESR Facility, The University
of Iowa.
§ Interdisciplinary Graduate Program in Human Toxicology, The University
of Iowa.
| University of Kentucky.
(1) Robertson, L. W.; Hansen, L. G. Recent AdVances in the EnVironmental
Toxicology and Health Effects of PCBs; University Press of Kentucky: Lexington,
2001.
(2) Hansen, L. G. The Ortho Side of PCBs: Occurrence and Disposition;
Kluwer Academic Publishers: Boston, 1999.
8296 J. Org. Chem. 2008, 73, 8296–8304
10.1021/jo801397g CCC: $40.75 2008 American Chemical Society
Published on Web 10/08/2008

Oxygenated PCBs: Synthesis and EPR properties
broad range of adverse biological effects, including (neuro-)-
developmental toxicity3,4 and carcinogenesis.5
apparent persistence of semiquinone radicals in the setting of
our experiments is a complicated result of the thermodynamic
or kinetic properties, or a combination, of all three redox forms,
i.e., Q, SQ•-, and H2Q. The work presented here does not
address this directly. However, it is clear that the yield of
semiquinone radical varies greatly among the (hydro-)quinones
studied; both thermodynamic and kinetics will play a role in
these apparent yields. Unfortunately the synthesis of oxygenated
PCB derivatives with differing degrees of chlorination of the
oxygenated ring system still represents a synthetic challenge.
PCB catechols, hydroquinones, and quinones have been tradi-
tionally synthesized using a variety of approaches, such as the
Cadogan14 and Ullmann coupling reactions.13 These reactions
typically have poor yields, require tedious cleanup procedures
and/or result in the formation of undesired15 and potentially toxic
byproducts.16,17 In addition, chlorinated starting materials with
suitable substitution patterns are frequently not available. Novel
synthetic strategies to overcome these difficulties are, therefore,
needed to make reactive PCB metabolites available for further
studies of the corresponding PCB quinones and semiquinone
radicals.
Mechanisms of PCB toxicity are still incompletely under-
stood. One reason is that commercial PCB products contain a
complex mixture of many of the possible 209 PCB derivatives
or congeners. Depending on the degree of chlorination and the
substitution pattern, PCB congeners have different modes of
action. In particular, environmentally persistent PCB congeners
interact with a number of cellular receptors and cause adverse
effects by interfering with inter- and intracellular signaling
processes and gene expression. Other PCB congeners, especially
lower chlorinated PCB congeners and congeners with vicinal
hydrogen atoms, are more rapidly metabolized2,6 and can form
reactive metabolites.7 These metabolites, which include PCB
epoxides, dihydroxy PCBs, and PCB quinones, have been shown
to react rapidly with cellular nucleophiles, such as glutathione,
amino acids, and DNA, in vitro and in vivo.7-10 A quinone
metabolite of 4-chlorobiphenyl (PCB 3), 2-(4-chlorophenyl)-
[1,4]benzoquinone, has also been implicated as the ultimate
carcinogen in the Solt-Faber protocol, an in vivo liver carcino-
genesis test.11
Here we report strategies for the synthesis of oxygenated PCB
congeners with differing degrees of chlorination (1 to 3 Cl
substituents in the oxygenated ring system). In addition, we
report an initial investigation of the semiquinone radicals formed
by these compounds using electron paramagnetic resonance. We
show that the yield of the PCB-derived semiquinone radicals
resulting from comproportionation of quinone and hydroquinone
depends on the oxygen and chlorine substitution pattern and
especially the number of chlorines on the semiquinone ring.
A recent electron paramagnetic resonance (EPR) study from
our laboratory demonstrated that lower chlorinated dihydroxy
PCBs and PCB quinones derived from PCB 3 and other PCB
congeners can form reactive PCB semiquinone free radicals by
autoxidation under basic conditions (pH > 7) as well as by
(enzymatic) one-electron transfer and comproportionation reac-
tions.12 This study provides direct evidence that PCB metabolites
can form reactive PCB semiquinone radicals, which are likely
to play an important but poorly understood role in the toxicity
of PCBs. PCB semiquinone radicals can facilitate the generation
of superoxide radicals (O2•-), which in the presence of super-
oxide dismutase forms hydrogen peroxide; H2O2 can lead to
the production of hydroxyl radicals (HO•). Subsequently, these
reactive oxygen species and, possibly, PCB semiquinone radicals
themselves can react with cellular nucleophiles, such as protein
and nonprotein sulfhydryls, resulting in oxidative stress.7
In addition to lower chlorinated PCBs, some higher chlori-
nated PCBs can be rapidly metabolized to mono- and dihy-
droxylated PCB metabolites.13 It is currently unclear how the
degree of chlorination in the metabolites of these higher
chlorinated PCBs alters the formation, persistence, and proper-
ties of the corresponding PCB semiquinone radicals. The
Results and Discussion
Synthesis of PCB Hydroquinones/Quinones with One
Chlorine Substituent on the Oxygenated Ring. Metabolism
of PCB congeners by cytochrome P-450 enzymes frequently
results in the formation of PCB metabolites with 2,5- and 3,4-
dihydroxy substitution patters.13,18 If no chlorine substituents
are present in the oxygenated ring system, these catechol and
hydroquinone metabolites can be easily synthesized, for ex-
ample, from 3-bromo-1,2-dimethoxy-benzene and a chlorinated
benzene boronic acid using the Suzuki coupling reaction.19-21
In contrast, the synthesis of PCB metabolites with a single
chlorine substituent in the oxygenated ring system is less
straightforward because suitable starting materials are not
available. Typically, the approaches used for the synthesis of
these metabolites result in product mixtures because of poor
selectivity and give the desired product in poor yields.13,14 We
investigated the selective chlorination of dimethoxy-benzenes
1 or 2 using H2O2/HCl to overcome these problems and to obtain
starting materials that can be used to synthesize putative
(3) Kodavanti, P. R. S. In Molecular Neurotoxicology: EnVironmental Agents
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EnVironmental Chemistry, Vol. 3 Part K. New Types of Persistent Halogenated
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Toxicology; OICA International: London, 2000; pp 16-32.
(8) Lin, P. H.; Sangaiah, R.; Ranasinghe, A.; Upton, P. B.; La, D. K.; Gold,
A.; Swenberg, J. A. Chem. Res. Toxicol. 2000, 13, 710–718.
(9) Arif, J. M.; Lehmler, H.-J.; Robertson, L. W.; Gupta, R. C. Chem.-Biol.
Interact. 2003, 142, 307–316.
(14) Lin, P.-H.; Sangaiah, R.; Ranasinghe, A.; Ball, L. M.; Swenberg, J. A.;
Gold, A. Org. Biomol. Chem. 2004, 2, 2624–2629.
(15) Shaikh, N. S.; Parkin, S.; Lehmler, H.-J. Organometallics 2006, 25,
4207–4214.
(16) Goldstein, J. A.; Hass, J. R.; Linko, P.; Harvan, D. J. Drug Metab.
Dispos. 1978, 6, 258–264.
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(11) Espandiari, P.; Glauert, H. P.; Lehmler, H.-J.; Lee, E. Y.; Srinivasan,
C.; Robertson, L. W. Toxicol. Sci. 2004, 79, 41–46.
(12) Song, Y.; Wagner, B. A.; Lehmler, H.-J.; Buettner, G. R. Chem. Res.
Toxicol. 2008, 21, 1359–1367.
(13) Waller, S. C.; He, Y. A.; Harlow, G. R.; He, Y. Q.; Mash, E. A.; Halpert,
J. R. Chem. Res. Toxicol. 1999, 12, 690–699.
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Toxicol. 1996, 9, 158–164.
(19) Lehmler, H.-J.; Robertson, L. W. Chemosphere 2001, 45, 137–143.
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Chemosphere 2004, 56, 735–744.
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Song et al.
SCHEME 1. Synthesis of PCB Hydroquinones/Quinones with One Chlorine on the Oxygenated Ring
metabolites of several lower chlorinated biphenyls using the
Suzuki coupling reaction.
observed with dimethoxy-benzene 5. These side reactions, which
are consistent with a radical mechanism, explain the lower yield
with the iodo compound 2. The yield of debrominated side
products (i.e., the respective chlorinated dimethoxy benzenes)
increased when an excess of H2O2/HCl was employed. Overall,
chlorination with H2O2/HCl is not a straightforward approach
for the preparation of bromo (or iodo) dimethoxy-benzenes with
two or three chlorine substituents because the impurities are
difficult to remove.
Chlorination of aromatic compounds with a H2O2/HCl system
was developed several decades ago and, for example, used to
fully chlorinate dimethoxy-benzene 2 for the synthesis of a
dihydroxy metabolite of PCB 136 (2,2′,3,3′,6,6′-hexachlorobi-
phenyl).13 However, controlling the regioselectivity of this
chlorination reaction appears to be a challenge,22 possibly
because this chlorination reaction is a radical reaction. Here we
report that dimethoxy-benzenes 1, 2, and 5 can be chlorinated
regioselectively with H2O2/HCl (Scheme 1A). The best yields
were obtained at molar ratios of dimethoxy-benzene/H2O2/HCl
of 1:4:8. However, the monochlorinated products 3, 4, and 6
were even isolated when an excess of chlorination reagent (8
equiv of HCl) was employed. The yields for iodo compound 2
(24%) were significantly lower compared to bromo compound
1 (71%).
The brominated dimethoxy-benzenes 3 and 6 were subse-
quently converted into dihydroxy compounds 9-10 and 13-14.
These compounds are putative metabolites of 2-chlorobiphenyl
(PCB 1), 2,4′-dichlorobiphenyl (PCB 8), 4-chlorobiphenyl (PCB
3), and 4,4′-dichlorobiphenyl (PCB 15), respectively (Scheme
1B). In the first step of the synthesis, the corresponding
dimethoxy PCBs were synthesized with the Suzuki coupling
of 3 and 6 with benzene or 4-chlorobenzene boronic acid.19-21
This cross-coupling reaction was highly efficient in the case of
4-chlorophenylboronic acid (98% yield for compounds 8 and
12), whereas lower yields were obtained for Suzuki coupling
reactions involving benzene boronic acid (66% and 71% for
compounds 7 and 11, respectively). These comparatively low
yields are probably due to the more difficult purification of lower
GC-MS analysis showed that monochlorination of dimethoxy-
benzenes 1 and 2 resulted in the formation of traces of 4,5-
dichloro-1,2-dimethoxy-benzene and 4,5-dibromo- (or 4,5-
diiodo-) 1,2-dimethoxy-benzene. Similar side products were
(22) Samant, B. S.; Saraf, Y. P.; Bhagwat, S. S. J. Colloid Interface Sci.
2006, 302, 207–213.
8298 J. Org. Chem. Vol. 73, No. 21, 2008

Oxygenated PCBs: Synthesis and EPR properties
FIGURE 1. Molecular structures derived from the crystal structure of 13 and 20. (a) Molecular structure of 4-chloro-biphenyl-2,5-diol (13) showing
the atom-labeling scheme and (b) view of 4-chloro-biphenyl-2,5-diol (13) along the C1-C1′ axis illustrating the nonplanar conformation of the
molecule. (c) Molecular structure of 3,6,4′-trichloro-biphenyl-2,5-diol (20) showing the atom-labeling scheme and (d) view of 3,6,4′-trichloro-
biphenyl-2,5-diol (20) along the C1-C1′ axis illustrating the nonplanar conformation of the molecule. Displacement ellipsoids are drawn at the
50% probability level.
chlorinated biphenyls with ortho substituents, consistent with
earlier observations from our laboratory.19-21 Subsequently, the
dimethoxy PCBs were demethylated with BBr3 in dichloro-
methane.20,21 Recrystallization using CHCl3/n-hexanes ) 1:3
(v/v) gave the desired PCB catechol and hydroquinone metabo-
lites in moderate-to-good yields. The only exception was
compound 9, which was relatively unstable at ambient
temperature.
The structure of PCB hydroquinone 13 was further character-
ized by single-crystal X-ray diffraction analysis (Figure 1a and
b). The solid state twist angle of compound 13, an important
structural determinant of the toxicity of PCBs and their
metabolites, is 43.3°.
PCB o-quinones are typically synthesized in situ by oxidation
of the corresponding catechols with Ag2O.11 This approach has
the drawback that PCB catechols themselves are unstable and
cannot be stored for extended periods of time without consider-
able degradation. Therefore, we explored the direct oxidation
of dimethoxy PCBs 8 or 12 to the corresponding PCB quinones
using CAN, a strong one-electron oxidizing reagent that can
be used as a reagent for a wide variety of organic transforma-
tions, including the oxidation of 1,4-hydroquinones and methoxy
compounds to the corresponding quinones.23 Unfortunately,
oxidation of compound 8 with CAN failed to yield the desired
PCB o-quinone as the major product and in a purity sufficient
for biological studies. However, the oxidation of dimethoxy PCB
12 with CAN gave the PCB p-quinone 15 in good yield (Scheme
1C).
stitution of bromine (or iodine) with chlorine. This approach
was abandoned because the resulting byproducts were tedious
to separate from the desired products. Chlorination of 1 and 5
with other chlorination reagents, such as NCS, was also not
successful. Therefore, we explored a different synthetic strategy
and investigated the introduction of a substituent suitable for
the Suzuki coupling reaction (e.g., bromine) into a dichlorinated
dimethoxy-benzene, such as 16. Although the bromination of
bulky and unreactive aromatic compounds is well documented
in the literature, the bromination of compound 16 proved to be
a challenge. Bromination of 16 with a series of bromination
reagents (such as Br2/AcOH,24 pyridinium bromide perbro-
mide,25 NBS26 and NBS/HBF4 ·Et2O27) did not yield the desired
product. Only Br2 in the presence of iron powder28 yielded a
brominated product in 43% yield (Scheme 2). Surprising, the
product was not the desired dimethoxy-benzene 18 but the
demethylated product 17. The dimethoxy-benzene 18 was
obtained by methylation with Me2SO4. Subsequently, the
dimethoxy PCB 19 was obtained in moderate yield by the
Suzuki coupling of 18 with 4-chlorobenzene boronic acid.
Demethylation with BBr3 gave the desired hydroquinone 20.
The structure of compound 20 was confirmed by single-crystal
X-ray diffraction analysis (Figure 1c and d). The solid state twist
angle of compound 20 is 73.2° and, because of the ortho
(24) Clive, D. L. J.; Khodabocus, A.; Vernon, P. G.; Angoh, A. G.; Bordeleau,
L.; Middleton, D. S.; Lowe, C.; Kellner, D. J. Chem. Soc., Perkin Trans. 1 1991,
1433–1444.
(25) Shimbashi, A.; Tsuchiya, A.; Imoto, M.; Nishiyama, S. Bull. Chem.
Soc. Jpn. 2004, 77, 1925–1930.
(26) Goldberg, Y.; Bensimon, C.; Alper, H. J. Org. Chem. 1992, 57, 6374–
6376.
(27) Oberhauser, T. J. Org. Chem. 1997, 62, 4504–4506.
(28) Liu, H.; Bernhardsen, M.; Fiksdahl, A. Tetrahedron 2006, 62, 3564–
3572.
Synthesis of PCB Hydroquinones/Quinones with Two
Chlorine Substituents on the Oxygenated Ring. Reaction of
1, 2, and 5 with excess of H2O2/HCl resulted in partial sub-
(23) Nair, V.; Deepthi, A. Chem. ReV. 2007, 107, 1862–1891.
J. Org. Chem. Vol. 73, No. 21, 2008 8299

Song et al.
SCHEME 2. Synthesis of PCB Hydroquinones/Quinones with Two Chlorines on the Oxygenated Ring
SCHEME 3. Synthesis of PCB Hydroquinones/Quinones with Three Chlorines on the Oxygenated Ring
chlorine substituent, larger compared to the twist angle of
compound 13. Oxidation of 19 with CAN yielded p-quinone
PCB 21 (Scheme 2). The oxidation of compounds 19 to 21 was
slower than the oxidation of the monochlorinated compounds
12 to 15 (1 vs 3 h), which suggests that multiple chlorine
substituents reduce the reaction rate of CAN oxidations.
Synthesis of PCB Hydroquinones/Quinones with Three
Chlorine Substituents on the Oxygenated Ring. We explored
the synthesis of PCB hydroquinones and quinones with three
chlorines on the oxygenated ring via the Suzuki coupling
reaction with the goal to exploit the selectivity and versatility
of this approach. The Suzuki coupling of a chlorinated (instead
of a brominated) benzene derivative with a boronic acid is
generally not suitable for the synthesis of PCB derivatives
because of the unselective coupling of all chlorine substituents.
However, the Suzuki coupling of symmetrical chlorinated
dimethoxy-benzenes, such as 2,3,5,6-tetrachloro-1,4-dimethoxy-
benzene 23, with benzene boronic acids can be used for the
synthesis of the desired dimethoxy biphenyls, such as 24
(Scheme 3A). The Suzuki coupling reaction of 23 with benzene
boronic acids using Pd(PPh3)4 with Na2CO3-toluene19-21 or
Pd(OAc)2 with di-tert-butylphosphino-biphenyl as ligand and
potassium phosphate as base did not yield the desired product
24. However, crude compound 24 (containing 12.5% of 2,3,5-
trichloro-1,4-dimethoxy-benzene) was obtained with Pd(OAc)2
in 32% yield using 2-dicyclohexylphosphino-2’,6’-dimethoxy-
biphenyl (DPDB) as the ligand and potassium phosphate as the
base.29-31 Subsequently, demethylation of crude 24 with
20,21
BBr3
yielded 3,4,6-trichloro-biphenyl-2,5-diol 25 in good
yield (Scheme 3A).
Several approaches were investigated for the synthesis of 2,3,5-
trichloro-6-phenyl-[1,4]benzoquinone 26, including CAN oxidation
of 24 (as described above), coupling of chloranil and benzene bor-
onic acid with Pd(OAc)2/2-dicyclohexylphosphino-2’,6’-dimethoxy-
biphenyl/potassium phosphate, and chlorination of biphenyl-2,5-
32
diol 27 with H2O2/HCl/FeCl3. However, the purification of 26
proved to be difficult because the compound rapidly degraded
during handling, even at subambient temperatures.
EPR Studies of PCB Semiquinones. Dihydroxylated aro-
maticcompounds,forexampledopamine,33 catecholestrogens,34,35
(29) Zapf, A.; Ehrentraut, A.; Beller, M. Angew. Chem., Int. Ed. 2000, 39,
4153–4155.
(30) Wolfe, J. P.; Buchwald, S. L. Angew. Chem., Int. Ed. 1999, 38, 2413–
2416.
(31) Wolfe, J. P.; Singer, R. A.; Yang, B. H.; Buchwald, S. L. J. Am. Chem.
Soc. 1999, 121, 9550–9561.
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8300 J. Org. Chem. Vol. 73, No. 21, 2008

Oxygenated PCBs: Synthesis and EPR properties
the nonoxygenated phenyl ring tend to exhibit hyperfine
splittings in the EPR spectra, but they can contribute to the line
width.12 Under identical experimental conditions (100 µM, pH
7.4), the autoxidation of 4-chloro-biphenyl-2,5-diol (13) gave
an approximate 1:2:1 three-line spectrum with aH3 ) 1.7 G and
aH6 ) 2.3 G (Figure 2b). Interestingly, the yield of SQ•- of 13
was 15-fold higher compared to SQ•- from 28, suggesting that
the electronegative chlorine substituent contributes to this
increase in the yield of SQ•-. As expected, compound 14, with
one chlorine substituent on each phenyl ring gives an ap-
proximate 1:2:1 three-line spectrum for its SQ•- (Figure 2c).
Interestingly, the intensity of this spectrum is about 30% greater
than the EPR spectrum for SQ•- from 13, again consistent with
the electronegative chlorine stabilizing SQ•-.
In contrast to SQ•- derived from PCB p-hydroquinones, the
EPR spectra of SQ•- formed by oxidation PCB o-hydroquinones
30 (4′-chloro-biphenyl-3,4-diol) and 9 are quite weak, yielding
an irregular spectrum12 and a 1:2:1 three-line spectrum (aH3
)
2.1 G and aH6 ) 2.1 G), respectively (Figure 2d and e).
Introducing a second chlorine such that both the oxygenated
and nonoxygenated rings have a chlorine (10) results in a
somewhat stronger EPR spectrum for the corresponding SQ•-
(1:2:1 three-line, aH3 ) 2.1 G and aH6 ) 2.1 G) compare Figure
2f to 2d and 2e. However, the apparent stabilizing effect of these
chlorines on the corresponding SQ•- is much less for PCB
o-SQ•- than PCB p-SQ•-. The greater steady-state concentration
for the PCB p-SQ•- compared to PCB o-SQ•- is most likely
due a slower rate of disproportionation for the p-SQ•-.
FIGURE 2. EPR spectra of semiquinones generated from PCB
hydroquinones with differing numbers and positions of chlorine on the
phenyl rings. Final concentrations of the original hydroquinones were
100 µM in phosphate buffer, pH ) 7.4. (a) SQ•- generated from 4′-
chloro-biphenyl-2,5-diol (28). (b) SQ•- generated from 4-chloro-
biphenyl-2,5-diol (13). (c) SQ•- generated from 4,4′-dichloro-biphenyl-
2,5-diol (14). (d) SQ•- generated from 4′-chloro-biphenyl-3,4-diol (30).
(e) SQ•- generated from 6-chloro-biphenyl-3,4-diol (9). (f) SQ•-
generated from 6,4′-dichloro-biphenyl-3,4-diol (10).
Because of the dramatic influence of chlorine on the
persistence of SQ•- derived from PCB p-hydroquinone (Figure
2b and c), we investigated the influence of additional chlorines
on the persistence of the corresponding SQ•-. Figure 3 shows
the EPR spectra of SQ•- formed by autoxidation of a series of
PCB p-hydroquinones and quinones with 0 to 3 chlorines on
the oxygenated phenyl ring; as the number of chlorines
increases, the persistence of the corresponding SQ•- increases.
As we have reported, the SQ•- formed from 4′-chloro-biphenyl-
2,5-diol (28) and 2-(4-chloro-phenyl)-[1,4]benzoquinone (29)
are essentially identical (Figure 3a and b).12 Similarly, the SQ•-
formed by PCB hydroquinones 14, 20, and 25 are identical to
the SQ•- formed by the corresponding PCB quinones 15, 21,
and 26, respectively. As anticipated, the spectra change with
increasing chlorine (and decreasing hydrogen) substitution from
1:3:3:1 four-line (28 and 29) to 1:2:1 three-line (14 and 15, aH3
or stilbene catechols,36 form reactive semiquinone radicals that
play an important role in their toxicity. Similarly, PCB hydro-
quinones, catechols, and quinones can form semiquinone
radicals12 that have been implicated as reactive intermediates
in PCB toxicity.9,37,38 Unfortunately, little is known about the
effects of chemical structure (e.g., substitution pattern and degree
of chlorination) on the properties of PCB semiquinone radicals
(SQ•-) and, ultimately, their toxicity. Here we investigate the
SQ•- formed from PCB hydroquinones and quinones with
varying numbers of chlorines on the oxygenated ring.
Autoxidation of the 2,5-dihydroxylated PCB metabolite 28
(4′-chloro-biphenyl-2,5-diol) resulted in a weak but distinct
approximate 1:3:3:1 four-line spectrum (aH3 ) aH4 ) 2.1 G
and aH6 ) 2.5 G at pH 7.412,39), Figure 2a. This spectrum is
typical for oxygenated PCB metabolites with no chlorine
substituent on the oxygenated phenyl ring.12,40 We have shown
previously that neither chlorine nor hydrogen substituents on
) 1.7 G and aH6 ) 2.3 G) to 1:1 two-line (20 and 21, aH4
)
1.9 G) to one-line spectra (25 and 26, no hyperfine splitting).
Another interesting phenomenon is that the g-factor of each of
the SQ•- changed, with the increasing of numbers of chlorine:
2.0047 (28 and 29); 2.0050 (14 and 15); 2.0051 (20 and 21);
and 2.0054 (25 and 26). This systematic increase in g-value is
consistent with the increased spin-orbit coupling that is
expected with increasing number of chlorines.
(33) Kalyanaraman, B.; Premovic, P. I.; Sealy, R. C. J. Biol. Chem. 1987,
262, 11080–11087.
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2267.
The most intriguing observation is the apparent exponential
increase in the initial yield of SQ•- with increasing degree of
chlorination in the oxygenated ring system (Figure 3). The
apparent yield of SQ•- increases from 0.000 092/0.000 12 for
28/29 to 0.23/0.23 for 25/26. Thus, as the degree of chlorination
increases, the persistence of the radical also increases. The three-
dimensional structure did not influence SQ•- persistence (e.g.,
the dihedral angle; Figure 1b and d). To examine this systemati-
cally, we generated each of the radicals shown in Figure 3 via
the comproportionation reaction of quinone and hydroquinone.
(40) Pedersen, J. A. Handbook of EPR Spectra from Quinones and Quinols;
CRC Press, Inc.: Boca Raton, FL, 1985.
J. Org. Chem. Vol. 73, No. 21, 2008 8301

Song et al.
Q + H2Q h 2SQ•- + 2H+
The expression for the equilibrium constant for this reaction
has the form K ) [SQ•-]2/[hydroquinone][quinone], assuming
constant pH. EPR spectra were collected and quantitated from
solutions with equal concentrations of PCB hydroquinone and
PCB quinone and the resulting concentrations used to estimate
the equilibrium constant for the comproportionation reaction
of each set of PCB hydroquinones and quinones of Figure 3.
As seen in Figure 4, the estimated equilibrium constant increases
by approximately 102.3 ) 200 with each addition of chlorine to
the oxygenated ring. It is quite amazing that nearly 64% of the
trichloro-hydroquinone/quinone equilibrium system (25/26) ex-
ists as the semiquinone radical. These observations demonstrate
the additive nature of the ring chlorines and the size of the MO
coefficients to the persistence of the corresponding radical.
In summary, in this work we have presented efficient and
high-yielding synthetic routes for the preparation of a series of
oxygenated PCB metabolites with differing degrees of chlorina-
tion. The synthesis of these compounds is straightforward. These
oxygenated PCBs readily react with oxygen or via compropor-
tionation to yield corresponding semiquinone free radicals. The
greater the number of chlorines on the oxygenated ring, the more
persistent the semiquinone radical.
Experimental Section
General Procedure for the Preparation of Chlorinated Bromo-
(or Iodo-)dimethoxy-benzenes 3, 4, and 6. 1-Bromo-4-chloro-2,5-
dimethoxy-benzene (6).13 Hydrogen peroxide (30%, 15 mL, 145
mmol) was added over 1 h to a rapidly stirred solution of 1-bromo-
2,5-dimethoxy-benzene 5 (7.6 g, 35 mmol) in CHCl3 (30 mL) and
concentrated HCl (24 mL, 280 mmol). After 16 h at room
temperature, the mixture was extracted with CHCl3 (3 × 30 mL).
The organic extracts were combined, washed with 5% NaHSO3
(25 mL), dried over Na2SO4, and filtered, and the solvent was
evaporated under reduced pressure. The product was purified by
column chromatography on silica gel with n-hexanes/CHCl3 ) 1:1
(v/v), yielding 5.7 g (69%) of 6 as white crystals. Mp 134-135
°C; 1H NMR (400 MHz, CDCl3) δ 7.12(s, 1H), 6.94(s, 1H), 3.85(s,
3H), 3.84(s, 3H); 13C NMR (100 MHz, CDCl3) δ 150.3, 149.6,
121.8, 117.4, 114.3, 109.6, 57.1, 57.0; MS m/z (relative intensity)
250(61, M•+), 235(7). Anal. Calcd for C8H8BrClO2: C, 38.18; H,
3.21. Found: C, 38.33; H, 3.03.
FIGURE 3. EPR spectra of semiquinones generated from PCB
hydroquinones and quinones with differing numbers of chlorine on the
oxygenated phenyl rings. (a) SQ•- generated from 1 mM of 4′-chloro-
biphenyl-2,5-diol (28) (yield: 0.000 092). (b) SQ•- generated from 1
mM of 2-(4-chloro-phenyl)-[1,4]benzoquinone (29) (yield: 0.000 12).
(c) Computer simulation of SQ•- with three hydrogens. (d) SQ•-
generated from 100 µM of 4,4′-dichloro-biphenyl-2,5-diol (14) (yield:
0.004 5). (e) SQ•- generated from 100 µM of 2-chloro-5-(4-chloro-
phenyl)-[1,4]benzoquinone (15) (yield: 0.003 5). (f) Computer simula-
tion of SQ•- with two hydrogens. (g) SQ•- generated from 10 µM of
3,6,4′-trichloro-biphenyl-2,5-diol (20) (yield: 0.026). (h) SQ•- generated
from 10 µM of 2,5-dichloro-3-(4-chloro-phenyl)-[1,4]benzoquinone (21)
(yield: 0.025). (i) Computer simulation of SQ•- with one hydrogen. (j)
SQ•- generated from 1 µM of 3,4,6-trichloro-biphenyl-2,5-diol (25)
(yield: 0.23). (k) SQ•- generated from 1 µM of 2,3,5-trichloro-6-phenyl-
[1,4]benzoquinone (26) (yield: 0.23). (l) Computer simulation of SQ•-
with no hydrogen. These spectra were collected using a modulation
amplitude of 1.0 Gauss. Lowering the modulation amplitude to 0.1
Gauss (for spectra d and e) or 0.01 Gauss (for spectra g, h, j, and k)
did not reveal any possible hyperfine splittings from the chlorines on
the semiquinone moiety.
General Suzuki Coupling Procedure A for the Synthesis of 7,
8, 11, 12, and 19. 4,4′-Dichloro-2,5-dimethoxy-biphenyl (12).19-21
Sodium carbonate (20 mL, 2 M aq) was added to a solution of
1-bromo-4-chloro-2,5-dimethoxy-benzene (6) (5.2 g, 20.0 mmol)
and Pd(PPh3)4 (0.72 g, 3% molar ratio) in toluene (80 mL). A
solution of a 4-chloro-phenylboronic acid (4.7 g, 30.0 mmol) in
ethanol/toluene/H2O ) 8:1:1 (40 mL total) was added slowly to
the mixture under a nitrogen atmosphere. The reaction mixture was
maintained at 80 °C for 12 h. Hydrogen peroxide (30%, 2.0 mL)
was added slowly to the warm reaction mixture to destroy unreacted
boronic acid. The mixture was stirred at room temperature for an
additional 4 h and diluted with diethyl ether (120 mL). The reaction
mixture was extracted once with NaOH (40 mL, 2 M aq) and three
times with water (40 mL). The organic phase was dried over
MgSO4, and the solvents were removed under reduced pressure.
Column chromatography over silica gel with n-hexanes/CHCl3 )
3: 1 (v/v) as eluent followed by recrystallization with n-hexanes/
CHCl3 ) 3: 1 (v/v) yielded 5.8 g (98%) of 12 as white solid. Mp
MHz, CDCl3) δ 150.7, 149.6, 136.3, 133.6, 131.0, 128.9, 128.5,
122.3, 115.2, 114.4, 57.2, 56.6; MS m/z (relative intensity) 282(88,
M•+), 267(43), 232(100), 217(30), 189(17), 161(11) 126(16). Anal.
Calcd for C14H12Cl2O2: C, 59.36; H, 4.27. Found: C, 59.24; H, 4.19.
General Demethylation Procedure for the Synthesis of 9, 10,
13, 14, 20, and 25. 4,4′-Dichloro-biphenyl-2,5-diol (14).20,21 Boron
tribromide (18.0 mL, 1 M in n-hexanes, 2.2 equiv) was added to a
solution of the 4,4′-dichloro-2,5-dimethoxy-biphenyl (12) (2.36 g,
8.3 mmol) in anhydrous dichloromethane (50 mL) under a nitrogen
atmosphere. The reaction mixture was stirred at room temperature
for 14 h, cooled with ice/salt, and hydrolyzed with an equal volume
of ice-cold water. The aqueous phase was extracted with dichlo-
romethane (2 × 50 mL). The combined organic layers were washed
with water (3 × 25 mL) and dried over MgSO4, and the solvent
was removed under reduced pressure. The product was purified by
column chromatography on silica gel with n-hexanes/ethyl acetate
1
123-124 °C; H NMR (400 MHz, CDCl3) δ 7.46-7.37(m, 4H),
7.02(s, 1H), 6.89(s, 1H), 3.89(s, 3H), 3.77(s, 3H); 13C NMR (100
(41) Yamazaki, I.; Piette, L. H. J. Am. Chem. Soc. 1965, 87, 986–989.
8302 J. Org. Chem. Vol. 73, No. 21, 2008

Oxygenated PCBs: Synthesis and EPR properties
Suzuki Coupling Procedure B for the Synthesis of 3,4,6-
Trichloro-2,5-dimethoxy-biphenyl (24).29-31 K3PO4 (4.25 g, 20
mmol), Pd(OAc)2 (44 mg, 2% molar ratio), and 2-dicyclohexy-
lphosphino-2′,6′-dimethoxybiphenyl (164 mg, 4% molar ratio) were
added to a solution of 1,2,4,5-tetrachloro-3,6-dimethoxy-benzene
(23) (2.62 g, 10.0 mmol) and phenylboronic acid (1.83 g, 15.0
mmol) in toluene (50 mL) under nitrogen atmosphere. The reaction
mixture was then stirred at 110 °C for 48 h. Hydrogen peroxide
(2.0 mL, 30%) was added slowly to the warm reaction mixture to
destroy unreacted boronic acid. The mixture was stirred at room
temperature for an additional 4 h and diluted with diethyl ether
(60 mL). After filtration, the solvent was extracted once with NaOH
(20 mL, 2 M aq) and three times with water (20 mL). The organic
phase was dried over MgSO4, and the solvents were removed under
reduced pressure. Column chromatography over silica gel with
n-hexanes/CHCl3 ) 3:1 (v/v) as eluent followed by recrystallization
with n-hexanes/CHCl3 ) 3:1 (v/v) yielded 1.03 g (32%) of crude
24 (contained 12.5% of 2,3,5-trichloro-1,4-dimethoxy-benzene) as
white solid. Mp 49-51 °C; 1H NMR (400 MHz, CDCl3)
δ7.50-7.41(m, AA′XX′ system, 3H), 7.33-7.27(m, AA′XX′
system, 2H), 3.93(s, 3H), 3.44(s, 3H); 13C NMR (100 MHz, CDCl3)
δ 151.8, 150.2, 135.6, 134.3, 130.1, 128.45, 128.41, 127.9, 127.2,
111.6, 61.0, 60.9; MS m/z (relative intensity) 316(100, M•+), 301(40,
M - CH3), 266(80, M - CH3Cl). Anal. Calcd for (C14H11Cl3O2 +
1/8C14H11Cl3O2): C, 51.80; H, 3.44. Found: C, 51.79; H, 3.38.
2,3,5-Trichloro-6-phenyl-[1,4]benzoquinone (26).32 Biphenyl-2,5-
diol (27) (2.0 g, 10.7 mmol) was suspended in glacial acetic acid
(30 mL), and concentrated HCl (20 mL), followed by anhydrous
FeCl3 (3.4 g), was added to the solution. Hydrogen peroxide (20
mL, 30%) was dropped slowly into the reaction mixture (20 min),
and the mixture was heated under reflux for an additional 1 h. The
mixture was filtered and extracted with CHCl3 (3 × 20 mL). The
combined organic phases were washed with saturated NaHCO3 (20
mL) and water (3 × 20 mL) and dried over MgSO4, and the solvents
were removed under reduced pressure. Recrystallization with
n-hexanes/ethyl acetate ) 4: 1 (v/v) yielded 2.8 g (91%) of 26 as
a yellow solid. Mp 94-95 °C (lit.32 112-114 °C); 1H NMR (400
MHz, CDCl3) δ 7.58-7.46(m, 3H), 7.40-7.29(m, 2H); 13C NMR
(100 MHz, CDCl3) δ 175.6, 171.9, 143.9, 141.6, 140.6, 140.5,
130.5, 130.3, 129.8, 128.5; MS m/z (relative intensity) 288(100,
[M + 2H]•+), 251(90, M - Cl), 233(52); HRMS(EI) calcd for
C12H5Cl3O2 m/z ([M]•+) 285.9355, found 285.9350.
FIGURE 4. The equilibrium constant for the comproportionation
reaction to form SQ•- increases with the number of chlorines on the
oxygenated ring. Assay conditions: quinone and hydroquinone mixture
in 100 mM PBS buffer, pH 7.4. (a) Zero chlorine, 50 µM of 28 and 50
µM of 29 yields [SQ•-]ss ) 63 nM. (b) One chlorine, 5 µM of 14 and
5 µM of 15 yields [SQ•-]ss ) 70 nM. (c) Two chlorines, 500 nM of 20
and 500 nM of 21 yields [SQ•-]ss ) 87 nM. (d) Three chlorines, 50
nM of 25 and 50 nM of 26 yields [SQ•-]ss ) 65 nM. K ) [SQ•-]2/
[hydroquinone][quinone]. This equilibrium constant will actually be
dependent on pH because the pKa’s for the hydroquinones will be above
pH or near 7, whereas the pKa of the semiquinone radicals will be
somewhat acidic; for example, the pKa of the semiquinone radical of
unsubstituted benzoquinone/hydroquinone is 4.25.41
) 1:1 (v/v) as eluent followed by recrystallization using n-hexanes/
CHCl3 ) 3: 1 (v/v) yielded 1.18 g (55%) of 14 as white solid. Mp
1
118-120 °C; H NMR (400 MHz, CDCl3) δ 7.47-7.38(m, 4H),
6.98(s, 1H), 6.91(s, 1H), 5.19(s, 1H), 4.75(s, 1H); 13C NMR (100
MHz, CDCl3) δ 146.4, 145.7, 134.7, 134.5, 130.5, 129.6, 127.6,
119.8, 117.2, 116.6; MS m/z (relative intensity) 398(47, [M +
TMS2]•+), 348(19), 239(13), 93(27), 73(100, [Si(CH3)3]+). Anal.
Calcd for C12H8Cl2O2: C, 56.48; H, 3.16. Found: C, 56.31; H, 3.12.
General Procedure for the Synthesis of PCB Quinones 15 and
21 from Methoxylated PCB Derivatives. 2-Chloro-5-(4-chloro-
phenyl)-[1,4]benzoquinone (15).42 4,4′-Dichloro-2,5-dimethoxy-
biphenyl 12 (0.54 g, 1.9 mmol) was dissolved in acetonitrile (10
mL) at 50 °C, a solution of CAN (5.8 g, 10.0 mmol) in water (15
mL) was added, and the reaction mixture was stirred at room
temperature for 1 h. The solution was extracted with chloroform
(2 × 20 mL) and washed with water (2 × 50 mL). The combined
organic phase dried over MgSO4, and the product was purified by
column chromatography on silica gel with CHCl3/n-hexanes ) 2:1
(v/v) as eluent, yielding 0.38 g (80%) of 15 as a yellow solid. Mp
EPR Spectroscopy. Electron paramagnetic spectroscopy was
performed using a Bruker EMX spectrometer equipped with a High
Sensitivity cavity and an Aqua-X sample holder. Spectra were
obtained at room temperature. Typical EPR-parameters were 3510.3
G center field; 15 G scan width; 9.854 GHz microwave frequency;
20 mW power; 2 × 105 receiver gain; modulation frequency of
100 kHz; modulation amplitude of 1.0 G; with the conversion time
and time constant both being 40.96 ms with 5 scans for each 1024-
point spectrum. Stock solutions (200 mM each) of PCB hydro-
quinones and quinones were prepared in DMSO. All experiments
were carried out in 100 mM phosphate buffer containing 250 µM
DETAPAC (diethylenetriaminepentaacetic acid) as described previ-
ously.12
137-139 °C (lit.43 147-148 °C); H NMR (400 MHz, CDCl3) δ
1
7.44(s, 4H), 7.10(s, 1H), 7.00(s, 1H); 13C NMR (100 MHz, CDCl3)
δ 184.3, 179.7, 145.4, 144.2, 137.3, 134.2, 132.3, 130.9, 130.5,
129.2; MS m/z (relative intensity) 254(30, [M + 2H]•+), 217(100).
Anal. Calcd for C12H6Cl2O2: C, 56.93; H, 2.39. Found: C, 56.84;
H, 2.35.
2-Bromo-3,6-dichloro-4-methoxy-phenol (17).28 A solution of
bromine (7.0 g, 44 mmol) in dichloromethane (10 mL) was added
slowly over 15 min to a solution of 1,4-dichloro-2,5-methoxy-
benzene 16 (8.5 g, 41 mmol) and iron powder (1.24 g, 22 mmol)
in dichloromethane (50 mL) heated under reflux. After 4 h, another
portion of iron powder (1.24 g, 22 mmol) was added and the
reaction heated for an additional 4 h. The reaction mixture was
allowed to cool to ambient temperature, the iron powder was filtered
off, and the product was purified by column chromatography on
silica gel with CHCl3/n-hexanes ) 2: 1 (v/v) as eluent to yield
Quantitation was accomplished by double integration of the
recorded EPR signals using the WinEPR program with 3-carbox-
yproxyl as the standard, ε234 ) 2370 ( 50 M-1 cm-1 44 To obtain
.
the best quantitative information on all samples and standards, they
were dissolved in identical aqueous buffer; after setup, the 4-bore
Aqua-X holder was not removed from the cavity for all samples.
Spectral simulations of EPR spectra were performed using the
WinSim program developed at the NIEHS by Duling.45 Correlation
coefficients of simulated spectra were typically >0.99.
1
4.8 g (43%) of 17 as white crystals. Mp 126-127 °C; H NMR
(400 MHz, CDCl3) δ 6.96(s, 1H), 5.60(s, 1H), 3.87(s, 3H); 13C
NMR (100 MHz, CDCl3) δ 150.2, 144.2, 123.4, 118.9, 113.1, 112.9,
57.4; MS m/z (relative intensity) 270(80, M+), 255(100), 227(20).
Anal. Calcd for C7H5BrCl2O2: C, 30.90; H, 1.85. Found: C, 30.84;
H, 1.73.
Acknowledgment. This research was supported by grants
ES05605, ES012475, and ES013661 from the National Institute
(42) Lopez-Alvarado, P.; Avendano, C.; Menendez, J. C. Synth. Commun.
2002, 32, 3233–3239.
(43) Bagli, J. F.; L’Ecuyer, P. Can. J. Chem. 1961, 39, 1037–1048.
(44) Venkataraman, S.; Martin, S. M.; Schafer, F. Q.; Buettner, G. R. Free
Radical Biol. Med. 2000, 29, 580–585.
J. Org. Chem. Vol. 73, No. 21, 2008 8303

Song et al.
of Environmental Health Sciences, NIH (H.-J.L. and L.W.R.)
and R01GM073929 from the National Institute of General
Medical Sciences (G.R.B.) and MRI grant 0319176 (S.P.) from
the National Science Foundation. Contents of this manuscript
are solely the reponsibility of the authors and do not necessarily
represent the official views of the NIEHS/NIH or NSF.
18-21, 23 and 25; 1H NMR and 13C NMR spectra for
compounds 3, 4, 6-15, 17-21, and 23-26; GC-MS chro-
matograms and MS spectra for compounds 9, 24, and 26;
crystallographic data for 13, 17, and 20 in CIF format. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO801397G
Supporting Information Available: Experimental proce-
dures, and characterization for compounds 3, 4, 7-11, 13,
(45) Duling, D. R. J. Magn. Res. Series B 1994, 104, 105–110.
8304 J. Org. Chem. Vol. 73, No. 21, 2008