2,2',3,3',6,6'-Hexachlorobiphenyl hydroxylation by active site mutants of cytochrome P450 2B1 and 2B11

Article abstract of DOI:10.1021/tx990030j

The structural basis of species differences in cytochrome P450 2B- mediated hydroxylation of 2,2',3,3',6,6'-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli. To facilitate metabolite identification, seven possible products, including three hydroxylated and four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of precursors and Ullmann and crossed Ullmann reactions. HPLC and GCfMS analysis and comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced 4,5-dihydroxy-236HCB and 5-hydroxy- 236HCB, while 2B11 L363V and 2B1 I114V mutants also catalyzed hydroxylation at the 4-position. The amount of products formed by 2B1 mutants I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L was smaller than that of the wild type. I477V exhibited unaltered 236HCB metabolism, and I480V produced twice as much dihydroxy product as the wild type. For 2B11, substitution of Val-114 or Asp-290 with Ile decreased the product yields. Replacement of Leu-363 with Val dramatically altered the profile of 236HCB metabolites. In addition to an increase in the overall level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4- position. Incubation of P450 2B1 with 5-hydroxy-236HCB produced 4,5- dihydroxy-236HCB, which indicates that 4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB. The findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367, 477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.

Full text of DOI:10.1021/tx990030j

690
Chem. Res. Toxicol. 1999, 12, 690-699
2,2′,3,3′,6,6′-Hexa ch lor obip h en yl Hyd r oxyla tion by Active
Site Mu ta n ts of Cytoch r om e P 450 2B1 a n d 2B11
Stephen C. Waller,†,‡,§ You Ai He,§,| Greg R. Harlow, You Qun He,|
Eugene A. Mash,† and J ames R. Halpert*,|
Department of Pharmacology and Toxicology, University of Texas Medical Branch,
Galveston, Texas 77555-1031, Synthetic Core Laboratory, Southwest Environmental Health Sciences
Center, Department of Chemistry, University of Arizona, Tucson, Arizona 85721, and Selectide,
a subsidiary of Hoechst Marion Roussel, 1580 East Hanley Boulevard, Tucson, Arizona 85737
Received February 24, 1999
The structural basis of species differences in cytochrome P450 2B-mediated hydroxylation
of 2,2′,3,3′,6,6′-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants
of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli. To
facilitate metabolite identification, seven possible products, including three hydroxylated and
four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of
precursors and Ullmann and crossed Ullmann reactions. HPLC and GC/MS analysis and
comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced
4,5-dihydroxy-236HCB and 5-hydroxy-236HCB, while 2B11 L363V and 2B1 I114V mutants
also catalyzed hydroxylation at the 4-position. The amount of products formed by 2B1 mutants
I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L
was smaller than that of the wild type. I477V exhibited unaltered 236HCB metabolism, and
I480V produced twice as much dihydroxy product as the wild type. For 2B11, substitution of
Val-114 or Asp-290 with Ile decreased the product yields. Replacement of Leu-363 with Val
dramatically altered the profile of 236HCB metabolites. In addition to an increase in the overall
level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4-position. Incubation
of P450 2B1 with 5-hydroxy-236HCB produced 4,5-dihydroxy-236HCB, which indicates that
4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB. The
findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367,
477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.
an important factor in the metabolism of PCBs, with the
rate of metabolism generally decreasing as the degree of
chlorination increases (9). The position of the chlorines
is also important, as demonstrated by Kato et al. (10) in
their work with four symmetrical hexachlorobiphenyl
isomers: 2,2′,3,3′,5,5′-HCB, 236HCB, 245HCB, and
2,2′,4,4′,6,6′-HCB. The authors observed that 236HCB,
which has adjacent unsubstituted carbons in the meta
and para positions, was much more rapidly metabolized
than the other three compounds. Interest in hydroxylated
PCBs has grown since it was recognized that these
compounds can possess greater estrogenicity or anties-
trogenicity than the parent PCBs (11, 12).
In tr od u ction
Polychlorinated biphenyls (PCBs)1 are ubiquitous en-
vironmental pollutants that accumulate in the environ-
ment due to their great lipophilicity and resistance to
biodegradation (1). PCBs have been detected in the
adipose tissue of humans and animals (2, 3). Certain
PCBs remain in the body for long periods of time,
resulting in a number of clinical manifestations such as
immune suppression, endocrine disruption, nervous sys-
tem effects, and especially hepatic dysfunction (4-6).
Mammalian metabolism of PCBs is catalyzed primarily
by cytochromes P450, the terminal oxidases of the hepatic
microsomal monooxygenase system (7, 8). The major
factor which determines whether a PCB will accumulate
in body tissue is its rate of hydroxylation to an excretable
form. The number of chlorines on the biphenyl ring is
Marked species differences exist in terms of the ability
to metabolize different PCB congeners. This provides an
excellent opportunity to examine the molecular basis of
species differences in substrate metabolism. For example,
245HCB and 236HCB are readily oxidized by dog liver
cytochrome P450 2B11, a member of the P450 2B
subfamily (13, 14). In contrast, the structurally related
cytochrome P450 in the rat (P450 2B1) oxidizes 245HCB
poorly, although rat P450 2B1 metabolizes 236HCB at a
much higher rate than 2B11 (13, 14). Neither compound
is a substrate for rabbit 2B4 or 2B5. A thorough under-
standing of enzyme function will require rigorous bio-
chemical analysis of both wild-type and mutant 2B
enzymes.
* To whom correspondence and reprint requests should be ad-
dressed. Telephone: (409) 772-9669. Fax: (409) 772-9642. E-mail:
jhalpert@UTMB.edu.
† University of Arizona.
‡ Current address: Bristol-Myers Squibb, 1 Squibb Dr., North
Brunswick, NJ 08903.
§ These authors contributed equally to this work.
| University of Texas Medical Branch.
Selectide.
1
Abbreviations: PCBs, polychlorinated biphenyls; HCB, hexa-
chlorobiphenyl; 236HCB, 2,2′,3,3′,6,6′-hexachlorobiphenyl; 245HCB,
2,2′,4,4′,5,5′-hexachlorobiphenyl; EtOAc, ethyl acetate; DLPC, dilau-
royl-L-3-phosphatidylcholine; HEPES, 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid.
10.1021/tx990030j CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/26/1999

2,2′,3,3′,6,6′-Hexachlorobiphenyl Metabolism
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 691
Sch em e 1. Syn th esis of 5-Hyd r oxy-236HCBa
Identifying the structural determinants of substrate
specificities of the cytochrome P450 2B subfamily has
been a major interest of our laboratory in recent years.
Our studies have provided evidence for an important role
for residues 114, 206, 209, 302, 363, 367, 477, 478, and
480 in 2B1 (15-19) and 114, 290, 363, and 480 in 2B11
(20, 21) in determining the regio- and stereoselectivity
of steroid metabolism. Mutants V114I,2 D290I, and
L363V in 2B11 also exhibited altered 245HCB metabolite
profiles (20), whereas L480V exhibited unaltered 245HCB
hydroxylase activity (21).
To delineate the structural features responsible for
236HCB hydroxylase activity, three mutants of 2B11 that
exhibited altered 245HCB metabolite profiles (20) and
14 available 2B1 mutants (15-19, 22) have been em-
ployed in this study and compared with wild-type 2B11
and 2B1. Only mutants L363V of 2B11 and I480V of 2B1
exhibited higher 236HCB hydroxylase activities than the
wild type. To be able to establish metabolite identity and
further study the toxic properties of 236HCB metabolites,
we report herein syntheses of monohydroxy and dihy-
droxy derivatives of 236HCB by methods that should
have broad applicability to syntheses of hydroxylated
derivatives of other PCBs. By comparison with authentic
compounds, the two metabolites produced by 2B1 and
2B11 wild type and all their mutants, and one new
metabolite produced only by 2B11 mutant L363V and
2B1 mutant I114V, were also identified in this study.
a
Details are given in Experimental Procedures.
4 h, the mixture was poured into saturated NaHCO3 (10 mL)
and extracted with CH2Cl2 (3 × 10 mL). The extracts were
combined, dried (Na2SO4), filtered, and concentrated. Prepara-
tive TLC (10% EtOAc/hexanes) and a single recrystallization
from 95% methanol/H2O gave 9 (2,2′,3,3′,6,6′-hexachloro-5-nitro-
1,1′-biphenyl, 34.0 mg, 0.084 mmol, 79%) as small white
crystals.
A mixture of 9 (36.8 mg, 0.091 mmol), absolute ethanol (3
mL), H2O (2 mL), and sodium hydrosulfite (86.6 mg, 0.423
mmol) was stirred at 80 °C for 4 h. After cooling to room
temperature, the mixture was poured into H2O (25 mL) and
extracted with Et2O (5 × 25 mL). These extracts were combined,
dried (MgSO4), filtered, and concentrated to a yellow solid.
Preparative TLC (20% EtOAc/hexanes) gave 10 (2,2′,3,3′,6,6′-
hexachloro-5-amino-1,1′-biphenyl, 14.8 mg, 0.039 mmol, 43%)
as a white solid.
Exp er im en ta l P r oced u r es
Sodium nitrite (4.0 mg, 0.058 mmol) was added to a solution
of 10 (10.6 mg, 0.028 mmol) in 50% H2SO4 (400 mL) at 0 °C.
After 10 min, the mixture was added via pipet in small portions
to boiling 66% H2SO4 (600 µL). After being refluxed for an
additional 10 min, this mixture was allowed to cool to room
temperature and was extracted with CH2Cl2 (3 × 0.5 mL). The
extracts were combined, dried (MgSO4), filtered, and concen-
trated. Preparative TLC (20% EtOAc/hexanes) gave 2 (5-
hydroxy-236HCB, 1.8 mg, 0.005 mmol, 18%), with an Rf of 0.29
(20% EtOAc/hexanes): 1H NMR (300 MHz, CDCl3) δ 5.74 (1, br
Ma ter ia ls. Organic starting materials for chemical syntheses
were purchased from Aldrich Chemical Co. (Milwaukee, WI).
Copper powder (40-100 mesh, 2N5) was purchased from Alfa
(Ward Hill, MA). Analytical TLC was performed using Merck
(Whitehouse Station, NJ ) 0.25 mm silica gel 60 F-254 plates.
Preparative TLC was performed using Merck 0.50 mm silica
gel 60 F-254 plates (20 cm × 20 cm). Gravity-driven column
chromatography was performed using Merck silica gel 60 (70-
230 mesh). Flash chromatography was performed using Merck
silica gel 60 (230-400 mesh). Growth media for Escherichia coli
were obtained from Difco (Detroit, MI). 236HCB (99%) was
obtained from Chem Service (West Chester, PA). [14C]236HCB
(2.44 dpm/pmol) was purchased from California Bionuclear (Sun
Valley, CA). NADPH and DLPC were purchased from Sigma
(St. Louis, MO). HEPES was obtained from Calbiochem Corp.
(La J olla, CA). Acetonitrile was purchased from J . T. Baker
(Phillipsburg, NJ ). All other reagents and supplies not listed
were obtained from standard sources. Ca u tion : PCBs and their
metabolites should be handled as hazardous compounds in
accordance with NIH guidelines (23).
Syn th eses of Meta bolites a n d Rela ted Com p ou n d s.
Reactions were carried out in vials exposed to the atmosphere,
except where otherwise noted. MS data were obtained at the
Southwest Environmental Health Sciences Center Analytical
Core Laboratory (Tucson, AZ). HRMS data were obtained in the
Mass Spectrometry Facility in the Department of Chemistry at
the University of Arizona. Elemental analyses were performed
by Desert Analytics (Tucson, AZ).
(1) 2,2′,3,3′,6,6′-Hexach lor o-5-h ydr oxy-1,1′-biph en yl [5-Hy-
d r oxy-236HCB (2), Sch em e 1]. A mixture of 97% H2SO4 (86
mg, 0.85 mmol) and 90% HNO3 (325 mg, 4.64 mmol) was added
in three portions over 8 h to a solution of 1 (24) (236HCB, 38.5
mg, 0.107 mmol) in CH2Cl2 (3 mL) at room temperature. After
s), 7.28 (1, s), 7.38 (1, d, J ) 9.0 Hz), 7.50 (1, d, J ) 9.0 Hz); 13
C
NMR (75 MHz, CDCl3) δ 117.9, 119.1, 124.2, 128.6, 131.2, 132.1,
132.7, 132.9, 133.3, 136.2, 136.4, 150.7; MS (70 eV) m/z (relative
intensity) 384 (1%), 382 (8%), 380 (36%), 378 (80%), 376 (100%),
374 (53%).
(2) 2,2′,3,3′,6,6′-Hexa ch lor o-5,5′-d ih yd r oxy-1,1′-bip h en yl
[5,5′-Dih yd r oxy-236HCB (5), Sch em e 2]. A mixture of 97%
H2SO4 (1.70 g, 16.8 mmol) and 90% HNO3 (5.26 g, 75.1 mmol)
was added to a solution of 1 (24) (236HCB, 470 mg, 1.30 mmol)
in CH2Cl2 (10 mL) at room temperature. After 1 h, the reaction
mixture was poured into saturated NaHCO3 (100 mL) and
extracted with CH2Cl2 (3 × 75 mL). The extracts were combined,
dried (Na2SO4), filtered, and evaporated to a thick yellow oil.
Flash chromatography on silica gel (75 g) eluted with hexanes
(200 mL) and 5% EtOAc/hexanes gave 11 (2,2′,3,3′,6,6′-
hexachloro-5,5′-dinitro-1,1′-biphenyl, 543 mg, 1.20 mmol, 92%)
as a white solid.
A mixture of 11 (510 mg, 1.1 mmol), absolute ethanol (20 mL),
H2O (5 mL), and sodium hydrosulfite (2.41 g, 11.8 mmol) was
stirred at 80 °C for 1 h. After cooling to room temperature, the
reaction mixture was poured into H2O (100 mL) and extracted
with CH2Cl2 (3 × 75 mL). The extracts were combined, dried
(Na2SO4), filtered, and evaporated to a white oily solid. Flash
chromatography on silica gel (75 g) eluted with 20% EtOAc/
hexanes gave 12 (2,2′,3,3′,6,6′-hexachloro-5,5′-diamino-1,1′-bi-
phenyl, 230 mg, 0.59 mmol, 54%) as a white solid.
2
Mutants are indicated using the single-letter code for the amino
acid residue that has been replaced, its position in the sequence, and
the single-letter designation of the new residue in the indicated order.
For example, V114I refers to replacement of Val at position 114 with
Ile.
To a solution of 12 (108 mg, 0.28 mmol) in distilled Et2O at
room temperature was added concentrated H2SO4 (40 µL, 0.72
mmol) dropwise. After 10 min, the solvent was evaporated, and

692 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Waller et al.
Sch em e 2. Syn th esis of 5,5′-Dih yd r oxy-236HCBa
mmol) was stirred at 80 °C for 2 h. The ethanol was removed
on a rotary evaporator, and H2O (100 mL) was added. The
resulting mixture was extracted with CH2Cl2 (2 × 100 mL).
These extracts were combined, dried (Na2SO4), filtered, and
evaporated to give a white solid. Recrystallization from ligroin
gave 15 (2,3,5-trichloro-6-methoxyaniline, 6.17 g, 27.3 mmol,
62%) as nearly colorless crystals.
Following the general procedure of Kajigaesi (26) for the
iodination of anisole, a mixture of 15 (2.87 g, 12.7 mmol), HOAc
(80 mL), benzyltrimethylammonium dichloroiodinate (4.74 g,
12.9 mmol), and ZnCl2 (3.17 g, 23.3 mmol) was stirred at room
temperature. After 18 h, H2O (100 mL) and 5% NaHSO3 (30
mL) were added, and the mixture was extracted with CH2Cl2
(3 × 100 mL). These extracts were combined, washed with
saturated NaHCO3 (3 × 100 mL), 5% NaHSO3 (100 mL), dried
(Na2SO4), filtered, and evaporated to give a red solid. Chroma-
tography on silica gel (100 g) eluted with hexanes (300 mL) and
5% EtOAc/hexanes gave 18 (2,3,5-trichloro-4-iodo-6-methoxya-
niline, 3.15 g, 8.94 mmol, 70%).
Isoamyl nitrite (3.50 mL, 25.3 mmol) was added dropwise to
a solution of 18 (641 mg, 1.82 mmol) and DMF (10 mL) at 75
°C. After 0.5 h, the reaction mixture was allowed to cool to room
temperature, poured into H2O (75 mL), and extracted with Et2O
(3 × 75 mL). These extracts were combined, dried (MgSO4),
filtered, and evaporated to give a red oil. Chromatography on
silica gel (75 g) eluted with 5% EtOAc/hexanes, followed by
recrystallization from pentane, gave 19 (2,4,5-trichloro-3-io-
doanisole, 287 mg, 0.85 mmol, 47%) as a white solid.
Following the general procedure of Hutzinger (27) for the
Ullmann coupling, a mixture of 19 (363.6 mg, 1.08 mmol) and
purified copper powder (373.9 mg, 5.88 mmol) in a sealed glass
tube flushed with argon was completely submerged in a sand
bath at 230 °C for 5 days. After the mixture cooled to room
temperature, the tube was opened and the contents were
extracted with CH2Cl2 (150 mL). The extracts were dried (Na2-
SO4), filtered, and evaporated to give a black solid. Flash
chromatography on silica gel (100 g) eluted with 2% EtOAc/
hexanes gave 20 (2,2′,3,3′,6,6′-hexachloro-5,5′-dimethoxy-1,1′-
biphenyl, 170 mg, 0.40 mmol, 74%) as a white solid.
A mixture of 20 (160 mg, 0.38 mmol), HOAc (10 mL), and
48% HBr (5.0 mL, 30 mmol) was heated at reflux for 40 h. After
cooling to room temperature, the mixture was poured into H2O
(50 mL) and extracted with CH2Cl2 (3 × 75 mL). The extracts
were combined, dried (Na2SO4), filtered, and evaporated to give
a yellow oil. Flash chromatography on silica gel (75 g) eluted
with 20% EtOAc/hexanes gave 5 (5,5′-dihydroxy-236HCB, 120
mg, 0.31 mmol, 82%) as a white solid, with an Rf of 0.12 (20%
EtOAc/hexanes): mp 110-112 °C; IR (Nujol) 3508, 1564, 1417,
1194, 1152 cm-1; 1H NMR (300 MHz, CDCl3) δ 5.70 (2, s), 7.28
(2, s); 13C NMR (75 MHz, CDCl3) δ 118.0, 119.0, 124.2, 132.8,
135.8, 150.7; MS (70 eV) m/z (relative intensity) 400 (3%), 398
(11%), 396 (35%), 394 (79%), 392 (100%), 390 (52%); HRMS m/z
calcd for C12H4Cl6O2 389.83425, found 389.83431 ( 0.00007.
Anal. Calcd for C12H4Cl6O2: C, 36.69; H, 1.03. Found: C, 36.63;
H, 1.04.
a
Details are given in Experimental Procedures.
50% H2SO4 (2.0 mL, 18 mmol) was added. This solution was
cooled to 0 °C, and solid sodium nitrite (82 mg, 1.2 mmol) was
added in small portions over 1 h. After 30 min, the cold yellow
solution was added dropwise to 66% sulfuric acid (3.0 mL, 36
mmol) maintained at 170-175 °C. After 15 min, the mixture
was allowed to cool to room temperature and was then extracted
with Et2O (5 × 5 mL). The extracts were combined and extracted
with 10% NaOH (2 × 10 mL) to give a red aqueous solution.
The aqueous extracts were combined, acidified using concen-
trated HCl (5 mL), and extracted with Et2O (4 × 20 mL). The
extracts were combined, dried (MgSO4), filtered, and evaporated.
Preparative TLC (20% EtOAc/hexanes) gave 5 (5,5′-dihydroxy-
236HCB, 6.8 mg, 0.017 mmol, 6%) as a white solid.
(3) 2,2′,3,3′,6,6′-Hexach lor o-4-h ydr oxy-1,1′-biph en yl [4-Hy-
d r oxy-236HCB (3)] a n d 2,2′,3,3′,6,6′-Hexa ch lor o-4,4′-d ih y-
d r oxy-1,1′-bip h en yl [4,4′-Dih yd r oxy-236HCB (6), Sch em e
3]. A mixture of 3,5-dichloroanisole (9.05 g, 51.1 mmol), CH2-
Cl2 (50 mL), 90% nitric acid (3.70 g, 52.8 mmol), and 97%
sulfuric acid (5.38 g, 53.2 mmol) was stirred at room tempera-
ture for 18 h. The reaction mixture was then partitioned
between H2O (50 mL) and CH2Cl2 (50 mL). The organic layer
was separated and washed with 1 N NaOH (100 mL). This basic
aqueous layer was extracted with CH2Cl2 (100 mL). The organic
phases were combined, dried (Na2SO4), filtered, and concen-
trated to give a yellow oil. This oil was dissolved in ethanol (90
mL), and H2O (20 mL) and sodium hydrosulfite (20.24 g, 0.10
mol) were added. This mixture was heated at reflux for 2 h.
After the mixture cooled to room temperature, the ethanol was
removed on a rotary evaporator, and H2O (50 mL) was added.
This mixture was extracted with Et2O (3 × 50 mL). The extracts
Alternatively as described by Harrison (25), 13 (2,4,5-trichlo-
roanisole, 10.16 g, 48.0 mmol) was added in small portions over
10 min to 90% nitric acid (27 mL, 0.64 mol) at 5 °C. The reaction
mixture was stirred at 5-10 °C for 45 min, poured into ice (100
g), and extracted with CH2Cl2 (2 × 100 mL). The extracts were
combined, dried (Na2SO4), filtered, and evaporated to give 14
(3,4,6-trichloro-2-nitroanisole, 11.37 g, 44.3 mmol, 92%) as an
orange oil.
A mixture of 14 (11.37 g, 44.3 mmol), absolute ethanol (100
mL), H2O (35 mL), and sodium hydrosulfite (37.65 g, 183.8

2,2′,3,3′,6,6′-Hexachlorobiphenyl Metabolism
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 693
(2,3,5-trichloro-4-iodoanisole, 422 mg, 1.25 mmol, 91%) as a
white solid.
Sch em e 3. Syn th esis of 4-Hyd r oxy- a n d
4,4′-Dih yd r oxy-236HCBa
A mixture of 28 (280 mg, 0.83 mmol), 21 (24) (1,2,4-trichloro-
3-iodobenzene, 260 mg, 0.84 mmol), and purified copper powder
(540 mg, 8.4 mmol) in a sealed glass tube under argon was
completely submerged in a sand bath at 230 °C for 6 days. After
cooling to room temperature, the contents were partitioned
between H2O (1 mL) and CH2Cl2 (50 mL). The organic layer
was separated, silica gel (1 g) added, and the solvent evaporated.
Flash chromatography on silica gel (75 g) eluted with 2% EtOAc/
hexanes gave
1 (236HCB, 53 mg, 0.15 mmol, 36%), 29
(2,2′,3,3′,6,6′-hexachloro-4-methoxy-1,1′-biphenyl, 111 mg, 0.28
mmol, 34%), and 30 (2,2′,3,3′,6,6′-hexachloro-4,4′-dimethoxy-1,1′-
biphenyl, 64 mg, 0.15 mmol, 36%) as white solids.
A mixture of 29 (104 mg, 0.27 mmol), HOAc (9 mL), and HBr
(48% in H2O, 4.5 mL, 27 mmol) was heated at reflux for 24 h.
After cooling to room temperature, the mixture was poured into
H2O (100 mL) and extracted with Et2O (3 × 100 mL). The
extracts were combined, dried (MgSO4), filtered, and concen-
trated to ca. 1.5 mL. Flash chromatography on silica gel (100
g) eluted with 5% EtOAc/hexanes gave 3 (4-hydroxy-236HCB,
59 mg, 0.16 mmol, 58%) as a white solid, with an Rf of 0.12
(10% EtOAc/hexanes): mp 147-150 °C; IR (Nujol) 3501, 1556,
1465, 1427, 1377, 1177, 975, 819 cm-1 1H NMR (250 MHz,
;
CDCl3) δ 5.89 (1, br s), 7.17 (1, s), 7.37 (1, d, J ) 8.7 Hz), 7.48
(1, d, J ) 8.7 Hz); 13C NMR (62.5 MHz, CDCl3) δ 115.6, 118.6,
128.4, 128.7, 131.0, 131.9, 133.0, 133.5, 133.7, 134.0, 136.5,
152.7; MS (70 eV) m/z (relative intensity) 384 (1%), 382 (9%),
380 (33%), 378 (80%), 376 (100%), 374 (51%); HRMS m/z calcd
for C12H4Cl6O 373.8393, found 373.8400 ( 0.0007. Anal. Calcd
for C12H4Cl6O: C, 38.24; H, 1.07. Found: C, 37.96; H, 0.89.
A mixture of 30 (47 mg, 0.11 mmol), HOAc (3.8 mL), and HBr
(48% in H2O, 1.9 mL, 11 mmol) was heated at reflux for 2 days.
After cooling to room temperature, the mixture was poured into
H2O (100 mL) and extracted with Et2O (4 × 75 mL). The extracts
were combined, dried (MgSO4), filtered, and evaporated to give
a yellow solid. Flash chromatography on silica gel (75 g) using
20% EtOAc/hexanes gave 6 (4,4′-dihydroxy-236HCB, 26 mg, 0.07
mmol, 64%) as a white solid, with an Rf of 0.08 (20% EtOAc/
hexanes): mp 175-177 °C; IR (Nujol) 3496, 1554, 1461, 1377,
a
Details are given in Experimental Procedures.
1
953 cm-1; H NMR (250 MHz, CDCl3) δ 5.89 (2, s), 7.16 (2, s);
13C NMR (62.5 MHz, CDCl3) δ 115.4, 118.5, 128.5, 133.7, 134.2,
152.7; MS (70 eV) m/z (relative intensity) 400 (2%), 398 (11%),
396 (35%), 394 (85%), 392 (100%), 390 (47%); HRMS m/z calcd
for C12H4Cl6O2 389.8343, found 389.8345 ( 0.0003. Anal. Calcd
for C12H4Cl6O2: C, 36.69; H, 1.03. Found: C, 37.20; H, 0.96.
(4) 2,2′,3′,4,6,6′-Hexa ch lor o-3-h yd r oxy-1,1′-bip h en yl (4)
a n d 2,2′,4,4′,6,6′-Hexa ch lor o-3,3′-d ih yd r oxy-1,1′-bip h en yl
(7) (Sch em e 4). A mixture of 2,4,6-trichloroanisole (3.30 g, 15.6
mmol), CH2Cl2 (100 mL), 90% HNO3 (5.09 g, 72.7 mmol), and
97% H2SO4 (2.53 g, 25.0 mmol) was stirred at room temperature
for 3 days. The mixture was carefully poured into saturated
NaHCO3 (150 mL) and extracted with CH2Cl2 (2 × 100 mL).
The extracts were combined, dried (Na2SO4), filtered, and
concentrated to give a red oil. Flash chromatography on silica
gel (75 g) using 10% EtOAc/hexanes gave 32 (2,4,6-trichloro-3-
nitroanisole, 1.73 g, 6.8 mmol, 44%) as an off-white solid.
A mixture of 32 (1.73 g, 6.8 mmol), ethanol (90 mL), H2O (20
mL), and sodium hydrosulfite (7.16 g, 35 mmol) was heated at
reflux for 4 h. After the mixture cooled to room temperature,
the solvent was evaporated and 10% EtOAc/hexanes (10 mL)
and silica gel (1 g) were added. This mixture was stirred for 10
min, and then the solvent was evaporated. Flash chromatog-
raphy on silica gel (75 g) eluted with 10% EtOAc/hexanes gave
33 (2,4,6-trichloro-3-methoxyaniline, 1.41 g, 6.2 mmol, 92%) as
a white solid.
To a solution of 33 (610 mg, 2.7 mmol) in Et2O (1.5 mL) was
added concentrated H2SO4 (160 mL, 2.9 mmol) dropwise. The
ether was evaporated, and 25% H2SO4 (8.0 mL, 36.0 mmol) was
added to the white solid. After the mixture cooled to 0 °C, a
solution of sodium nitrite (560 mg, 8.2 mmol) in H2O (9 mL)
was added over the course of 1 h. After an additional 30 min at
were combined, dried (MgSO4), filtered, and evaporated to give
a brown oil. Flash chromatography on silica gel (100 g) eluted
with 5% EtOAc/hexanes gave starting material 22 (3,5-dichlo-
roanisole, 3.48 g, 19.7 mmol, 38%) and the products 25 (2,6-
dichloro-4-methoxyaniline, 0.66 g, 3.4 mmol, 7%) as a white solid
and 26 (2,4-dichloro-6-methoxyaniline, 1.82 g, 9.5 mmol, 19%)
as a brown oil that decomposed at room temperature.
To a solution of 25 (580 mg, 3.0 mmol) in Et2O (5 mL) was
added concentrated H2SO4 (200 mL, 3.6 mmol) dropwise. The
ether was evaporated, and 25% H2SO4 (8.0 mL, 36.0 mmol) was
added to the white solid. After the mixture cooled to 0 °C, a
solution of sodium nitrite (0.42 g, 6.1 mmol) in H2O (6.2 mL)
was added over the course of 20 min. After an additional 10
min at 0 °C, a solution of potassium iodide (1.03 g, 6.2 mmol)
in H2O (10 mL) was added at 0 °C over the course of 5 min. The
resulting rust-colored solution was stirred at 0 °C for 4 h, and
at room temperature for 14 h, and was heated at reflux for 2 h.
After cooling to room temperature, the mixture was extracted
with Et2O (3 × 25 mL). The extracts were combined, washed
with 5% sodium bisulfite (20 mL), dried (MgSO4), filtered, and
concentrated to give a red oil. Flash chromatography on silica
gel (75 g) eluted with 5% EtOAc/hexanes gave 27 (3,5-dichloro-
4-iodoanisole, 510 mg, 1.7 mmol, 57%) as a white solid.
A mixture of 27 (416 mg, 1.37 mmol), HOAc (7 mL),
concentrated HCl (0.46 mL, 5.5 mmol), and 30% H2O2 (150 mL,
1.5 mmol) was stirred at room temperature for 16 h. Water (20
mL) was added, and the mixture was extracted with CH2Cl2 (3
× 25 mL). The extracts were combined, dried (Na2SO4), filtered,
and concentrated to give a white solid. Flash chromatography
on silica gel (100 g) eluted with 3% EtOAc/hexanes gave 28

694 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Waller et al.
Sch em e 4. Syn th esis of 3-Hyd r oxy-2,2′,3′,4,6,6′-HCB
a n d 3,3′-Dih yd r oxy-2,2′,4,4′,6,6′-HCBa
Sch em e 5. Syn th esis of 4,5-Dih yd r oxy-236HCBa
a
Details are given in Experimental Procedures.
After cooling to room temperature, the mixture was concen-
trated to give a yellow solid. Flash chromatography on silica
gel (50 g) using 20% EtOAc/hexanes gave 7 (2,2′,4,4′,6,6′-
hexachloro-3,3′-dihydroxy-1,1′-biphenyl, 48.4 mg, 0.123 mmol,
96%) as a white solid, with an Rf of 0.09 (20% EtOAc/hexanes):
mp 130-132 °C; IR (Nujol) 3516, 1462, 1377, 1194, 723 cm-1
;
1H NMR (300 MHz, CDCl3) δ 5.97 (2, br s), 7.47 (2, s); 13C NMR
(75 MHz, CDCl3) δ 121.8, 121.9, 125.4, 128.5, 133.6, 147.3; MS
(70 eV) m/z (relative intensity) 400 (1%), 398 (9%), 396 (35%),
394 (81%), 392 (100%), 390 (54%); HRMS m/z calcd for C12H4-
Cl6O2 389.8343, found 389.8344 ( 0.0002. Anal. Calcd for C12H4-
Cl6O2: C, 36.69; H, 1.03. Found: C, 36.85; H, 0.89.
(5) 2,2′,3′,5,6,6′-Hexa ch lor o-3,4-d ih yd r oxy-1,1′-bip h en yl
[4,5-Dih yd r oxy-236HCB (8), Sch em e 5]. Hydrogen peroxide
(30%, 10.0 mL, 97.8 mmol) was added over the course of 1 h to
a rapidly stirred mixture of 4-iodoveratrole 37 (27) (1.10 g, 4.2
mmol), CHCl3 (10 mL), and concentrated HCl (30 mL, 0.36 mol).
After 14 h at room temperature, the mixture was extracted with
CHCl3 (3 × 40 mL). The extracts were combined, washed with
5% NaHSO3 (50 mL), dried (Na2SO4), filtered, and evaporated.
Flash chromatography on silica gel (75 g) eluted with 2% EtOAc/
hexanes gave 38 (1,2,4-trichloro-3-iodo-5,6-dimethoxybenzene,
560 mg, 1.5 mmol, 36%) as a white solid.
A mixture of 38 (290 mg, 0.79 mmol), 21 (24) (1,2,4-trichloro-
3-iodobenzene, 270 mg, 0.88 mmol), and purified copper powder
(580 mg, 9.1 mmol) in a sealed, glass tube under argon was
completely submerged in a sand bath at 230 °C for 7 days. After
the mixture cooled to room temperature, CH2Cl2 (50 g) and silica
gel (1 g) were added, and the solvent was evaporated. Flash
chromatography on silica gel (75 g) eluted with 2% EtOAc/
hexanes gave 1 (236HCB, 61 mg, 0.17 mmol, 39%) and 39
(2,2′,3′,5,6,6′-hexachloro-3,4-dimethoxy-1,1′-biphenyl, 30 mg,
0.07 mmol, 9%) as an oil.
A mixture of 39 (21.6 mg, 0.051 mmol), HOAc (4 mL), and
48% HBr (2.0 mL, 12 mmol) was heated at reflux for 2 days.
After cooling to room temperature, the mixture was concen-
trated to give a white solid. Preparative TLC using 40% EtOAc/
hexanes gave 8 (4,5-dihydroxy-236HCB, 12.3 mg, 0.031 mmol,
61%) as a white solid, with an Rf of 0.18 (40% EtOAc/hexanes):
1H NMR (500 MHz, CDCl3) δ 5.87 (2, br s), 7.36 (1, d, J ) 8.6
Hz), 7.47 (1, d, J ) 8.6 Hz); 13C NMR (125 MHz, CDCl3) δ 118.72,
118.96, 124.19, 127.82, 128.45, 131.07, 132.03, 133.71, 134.05,
136.26, 139.58, 141.59; MS (70 eV) m/z (relative intensity) 400
(1%), 398 (8%), 396 (39%), 394 (77%), 392 (100%), 390 (49%);
HRMS m/z calcd for C12H4Cl6O2 389.8343, found 389.8344 (
0.0002.
Heter ologou s Exp r ession . E. coli Topp3 cells were used
for P450 expression. Methods for expression and solubilization
of membranes were as described previously (28), except that
the P450 concentration in sonicated whole cells was measured
daily to identify peak expression levels before membrane
preparation.
a
Details are given in Experimental Procedures.
0 °C, a solution of potassium iodide (1.64 g, 9.9 mmol) in H2O
(10 mL) was added over the course of 5 min. This was stirred
at 0 °C for 1 h and then heated at reflux for 16 h. After the
mixture cooled to room temperature, 5% sodium bisulfite (25
mL) was added, and this mixture was extracted with Et2O (3 ×
50 mL). The extracts were combined, dried (MgSO4), filtered,
and evaporated to give a yellow solid. Flash chromatography
on silica gel (50 g) eluted with hexanes gave 34 (2,4,6-trichloro-
3-iodoanisole, 740 mg, 2.2 mmol, 81%) as a white solid.
A mixture of 34 (240 mg, 0.71 mmol), 21 (24) (1,2,4-trichloro-
3-iodobenzene, 0.22 g, 0.71 mmol), and purified copper powder
(460 mg, 7.2 mmol) in a sealed, glass tube under argon was
completely submerged in a sand bath at 230 °C for 23 days.
After the mixture cooled to room temperature, CH2Cl2 (50 g)
and silica gel (1 g) were added, and the solvent was evaporated.
Flash chromatography on silica gel (75 g) eluted with hexanes
(200 mL) and 4% EtOAc/hexanes gave 1 (236HCB, 41 mg, 0.11
mmol, 31%), 35 (2,2′,3′,4,6,6′-hexachloro-3-methoxy-1,1′-biphe-
nyl, 63 mg, 0.16 mmol, 23%), and 36 (2,2′,4,4′,6,6′-hexachloro-
3,3′-dimethoxy-1,1′-biphenyl, 72 mg, 0.17 mmol, 46%) as white
solids.
A mixture of 35 (58.6 mg, 0.150 mmol), HOAc (5 mL), and
48% HBr (2.5 mL, 15 mmol) was heated at reflux for 16 h. After
cooling to room temperature, the mixture was poured into H2O
(25 mL) and extracted with CH2Cl2 (4 × 50 mL). The extracts
were combined, dried (Na2SO4), filtered, and concentrated to
give a white solid. Flash chromatography on silica gel (25 g)
using 5% EtOAc/hexanes gave 4 (2,2′,3′,4,6,6′-hexachloro-3-
hydroxy-1,1′-biphenyl, 54.7 mg, 0.145 mmol, 97%) as a white
solid, with an Rf of 0.13 (15% EtOAc/hexanes): mp 75-77 °C;
IR (Nujol) 3534, 1462, 1376, 1178, 811, 718 cm-1; 1H NMR (300
MHz, CDCl3) δ 5.92 (1, s), 7.38 (1, d, J ) 8.8 Hz), 7.47 (1, s),
7.49 (1, d, J ) 8.8 Hz); 13C NMR (75 MHz, CDCl3) δ 121.6, 121.8,
125.3, 128.5, 128.5, 131.2, 132.0, 133.1, 133.5, 134.4, 135.9,
147.3; MS (70 eV) m/z (relative intensity) 384 (1%), 382 (9%),
380 (35%), 378 (78%), 376 (100%), 374 (51%); HRMS m/z calcd
for C12H4Cl6O 373.8393, found 373.8400 ( 0.0007. Anal. Calcd
for C12H4Cl6O: C, 38.24; H, 1.07. Found: C, 38.46; H, 0.83.
A mixture of 36 (53.8 mg, 0.128 mmol), HOAc (4 mL), and
48% HBr (2.0 mL, 12 mmol) was heated at reflux for 2 days.
In cu ba tion Con d ition s. The reconstituted system contained
100 pmol of P450, 200 pmol of rat liver NADPH-cytochrome
P450 reductase (29), 30 µg/mL DLPC, and 200 pmol of rat liver
cytochrome b5 (30) and was incubated for 10 min at room

2,2′,3,3′,6,6′-Hexachlorobiphenyl Metabolism
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 695
temperature. DLPC (30 µg/mL) in 50 mM HEPES (pH 7.6), 15
mM MgCl2, and 0.1 mM EDTA was prepared and added to the
reconstituted enzymes. 236HCB (66 µM final concentration) was
added, and the reaction mixture was preincubated for 3 min at
37 °C. The final 1 mL reaction was started by adding NADPH
(1 mM final concentration), the mixture incubated at 37 °C for
1 h, and then the reaction terminated by acidification with 60
µL of 1 N H2SO4. The hydroxylated 236HCB metabolites were
extracted three times with 3 mL of HPLC-grade EtOAc. The
combined EtOAc extracts were evaporated to dryness under a
stream of nitrogen at 37 °C prior to further analysis using
HPLC.
necessary to establish the position of nitration. NMR
analysis was not useful for this purpose, and we were
unable to grow crystals of any of the products that were
suitable for X-ray crystallographic analysis. To ascertain
the regiochemistry of nitration, a second synthesis of 5
(5,5′-dihydroxy-236HCB) via an Ullmann coupling reac-
tion was undertaken (Scheme 2). 2,4,5-Trichloroanisole
(13) was nitrated following a literature procedure (25)
to give one regioisomer as the major product. However,
proof of the structure of this nitration product was not
previously presented. After reduction to the correspond-
ing anisidine using sodium dithionite, the position of the
amine group was established by demethylation and
condensation with benzoic acid to afford benzoxazole 17.
Thus, nitration of 13 had produced 14, which yielded 15
upon reduction and 16 upon demethylation. Anisidine 15
was iodinated using benzyltrimethylammonium dichlor-
oiodinate to give 18. Deamination of 18 via the diazonium
salt afforded iodoanisole 19. Coupling of 19 using purified
copper powder in a sealed tube at 230 °C gave dimethoxy
PCB 20. Demethylation of 20 gave a biphenol product
identical to 5 obtained via dinitration of 1.
The success of the synthesis of 5 via an Ullmann
coupling led to pursuit of other hydroxylated PCB deriva-
tives by this method. It occurred to us that crossed
Ullmann reactions involving different iodobenzene com-
ponents might lead to separable mixtures of parent,
monohydroxy, and dihydroxy PCBs in acceptable yields.
As shown in Scheme 3, to obtain the required iodoanisole
28, commercially available 3,5-dichloroanisole (22) was
nitrated to give a symmetrical product 23 and unsym-
metrical product 24. Separation of these products was
tedious, and so the product mixture was reduced with
sodium dithionite to give the separable anisidines 25 and
26. A Sandmeyer reaction of 25 using potassium iodide
gave iodide 27. Monochlorination of 27 proceeded smoothly
to give 28. Ullmann coupling of 28 with an equimolar
amount of 21 gave an easily separated mixture of 1, 29,
and 30. Demethylation of 29 and 30 gave the desired
hydroxylated PCBs 3 (4-hydroxy-236HCB) and 6 (4,4′-
dihydroxy-236HCB), respectively, through an unambigu-
ous synthetic route. A crossed Ullmann reaction was also
used to simultaneously prepare monohydroxy PCB de-
rivative 4 and dihydroxy PCB derivative 7 (Scheme 4).
A final example of this method involved the synthesis
of the unsymmetrical diol 8 (4,5-dihydroxy-236HCB,
Scheme 5).
Detection a n d Isola tion of 236HCB Meta bolites.
Figure 1A shows the HPLC chromatogram of the two
metabolites (M-1 and M-2) produced by E. coli-expressed
P450 2B1. These two peaks were not detected in the
incubation mixture in the absence of NADPH. P450 2B11
wild type and most of the 2B1 and 2B11 mutants formed
the same metabolites. P450 2B11 mutant L363V pro-
duced three metabolites, M-1, M-2, and a novel metabo-
lite M-3 (Figure 1B). The 2B1 mutant I114V also pro-
duced M-3 (data not shown). Interestingly, the M-1:M-2
ratio varied with the amount of P450 2B1 wild type, the
236HCB concentration, and the P450:reductase ratio in
the final reaction mixture (data not shown). The data
suggested that M-1 might be derived from M-2. There-
fore, the three metabolites were collected from HPLC for
initial GC/MS analysis. The trimethylsilylated M-1 gave
a molecular ion peak at m/z 534 and isotope peaks that
can be attributed to the six chlorine atoms in the
molecule, indicating that M-1 represented a dihydroxy-
Isola tion of 236HCB Meta bolites by HP LC. The dried
residue was redissolved in a mobile phase and transferred to a
1.5 mL microfuge tube. The redissolved mixture was centrifuged
to sediment any particulates, and injected into the HPLC
system. For HPLC analysis, a Zorbax RX-C8 column (5 µm ×
250 mm × 4.6 mm, Mac-Mod Analytical Inc., Chadds Ford, PA)
was used with a Brownlee RP-8 guard column (Rainin, Woburn,
MA). The UV detector was set at 298 nm. The metabolites were
eluted isocratically with a mobile phase of 50% component A
[0.05% (v/v) trifluoroacetic acid in HPLC-grade acetonitrile]/50%
component B [0.1% (v/v) trifluoroacetic acid in filtered Millipore
water] at a rate of 1.5 mL/min. For metabolite quantification
using radiolabled substrate, 30 s fractions were collected and
the amount of 14C in each fraction was determined in a Beckman
LS8100 liquid scintillation counter. The data were used to
determine the ratio of the picomoles of product (from specific
activity of the radiolabel) to peak area (from UV absorbance)
for each metabolite. These ratios were used to convert peak
areas to picomoles of metabolite for incubations with nonlabeled
substrate.
Id en tifica tion of 236HCB Meta bolites by GC/MS. Prior
to GC/MS analyses, 236HCB metabolites were separated by
HPLC. The metabolites were trimethylsilylated by N-methyl-
N-(trimethylsilyl)trifluoroacetamide or methylated by ethereal
diazomethane (Analytical Core, Southwest Environmental Health
Science Center). Dried metabolites were dissolved in EtOAc, and
1 µL of the sample was used for product identification on a Fison
MD-800 benchtop quadrupole GC/MS system. Products were
introduced by splitless injection and separated on a 30 m
DB5MS fused-silica capillary column (0.25 mm inside diameter,
0.25 µm film). High-purity helium was used as the carrier gas
at a head pressure of 10 psi. The temperature of the column
was held at 100 °C for 3 min, raised to 280 °C at rate of 15
°C/min, and maintained at 280 °C for 10 min. MS analysis was
carried out by electron ionization at 70 eV.
Resu lts
Syn th esis of 236HCB Meta bolites. Three methods
for the synthesis of these hydroxylated PCBs were
examined. Coupling of chlorinated diazonium salts with
O-protected chlorinated phenol derivatives by the method
of Cadogan (31, 32) was abandoned since, in our hands,
complex mixtures and low chemical yields were obtained.
A further issue was the difficulty in establishing the
structures of the purified products. A second synthetic
option involved direct functionalization of PCBs (33-37).
Reaction of 236HCB with nitric acid and sulfuric acid in
CH2Cl2 gave one mononitration product in good yield.
Subsequent work (vide infra) established that the nitra-
tion had occurred at position 5 to yield 9 (Scheme 1).
Reduction of 9 with sodium dithionite yielded aniline 10,
which was converted to phenol 2 (5-hydroxy-236HCB) via
the corresponding diazonium salt, albeit in low yield. The
dihydroxylated 236HCB at positions 5 and 5′ could be
similarly prepared (Scheme 2).
While chlorination of 236HCB had previously been
shown to be regioselective at position 5 (24), it was

696 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Waller et al.
F igu r e 2. Mass spectra of trimethylsilylated M-1 (compound
8, top panel), methylated M-2 (compound 2, middle panel), and
methylated M-3 (compound 3, bottom panel). GC/MS conditions
are described in Experimental Procedures.
To confirm the identity of M-1-M-3, the metabolites
from incubations with P450 2B1 and 2B11 mutant L363V
were collected from HPLC using the mobile phase of 50%
component A and 50% component B, which yielded
separation of M-2 and M-3. GC analysis was performed
by comparing the retention times of their derivatives and
spiking the metabolite derivatives with those of corre-
sponding standards. Identification of M-1 as 4,5-dihy-
droxy-236HCB was further confirmed by comparing the
fragmentation pattern of the trimethylsilyl derivative
(Figure 2) with that of authentic 4,5-dihydroxy-236HCB
(not shown). The trimethylsilyl derivatives of M-2 and
M-3 exhibited different retention times on GC, which
were identical to those of 5-hydroxy-236HCB and 4-hy-
droxy-236HCB standards, respectively. However, the two
standards could not be distinguished from each other
from their fragmentation patterns in mass spectra.
Therefore, M-2, M-3, and the standards were methylated
by ethereal diazomethane as described by Schnellmann
et al. (38). The methylated M-2 and M-3 exhibited
retention times on GC identical to those of their corre-
sponding standards. The mass spectra of the methylated
derivatives of M-2 and M-3 are shown in Figure 2. The
molecular ion was present at m/z 388 for both metabo-
lites. The mass spectra of the 5-methoxy and 4-methoxy
standards contained a significant peak at m/z 345 [M-43],
which results from the loss of CH3CO, and m/z 275
[M-113], which was probably formed by successive loss
of CH3CO and two chlorines. However, only the 5-meth-
oxy isomer gave the peak at m/z 373 [M-15], which
resulted from the loss of a methyl radical. Thus, M-2,
which had the peak at m/z 373 in the mass spectra of its
methylated derivative, was identified as 5-hydroxy-
F igu r e 1. HPLC chromatograms of 236HCB metabolites (A)
P450 2B1 wild type and (B) the 2B11 L363V mutant. M1 is 4,5-
dihydroxy-236HCB, M2 5-hydroxy-236HCB, and M3 4-hydroxy-
236HCB. HPLC conditions were the same as those described
in Experimental Procedures.
HCB. The trimethylsilylated derivatives of both M-2 and
M-3 gave a molecular ion peak at m/z 446 and isotopic
distribution of six chlorine atoms, indicating that the two
metabolites represented monohydroxy HCBs.
Id en tifica tion of 236HCB Meta bolites. The seven
authentic standards were separated by HPLC using the
mobile phase of 58% component A and 42% component
B. The retention times of M-1-M-3 were identical to
those of 4,5-dihydroxy-236HCB, 5-hydroxy-236HCB, and
4-hydroxy-236HCB, respectively. The metabolites were
then spiked with the three standards. The predicted
increase in the peak areas of metabolites and coelution
of the standards with the metabolites demonstrated that
M-1-M-3 may be 4,5-dihydroxy-236HCB, 5-hydroxy-
236HCB, and 4-hydroxy-236HCB, respectively.

2,2′,3,3′,6,6′-Hexachlorobiphenyl Metabolism
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 697
Ta ble 1. Meta bolism of 236HCB by P 450 2B11 a n d
Mu ta n tsa
amount of product (nmol h-1 100 pmol of P450-1
)
4,5-dihydroxy-236HCB
(% of 2B11 activity)
5-hydroxy-236HCB
(% of 2B11 activity)
2B11
0.72
0.30
V114I
D290I
L363Vb
0.30 (45)
0.36 (51)
1.08 (153)
0.24 (80)
0.30 (100)
0.60 (200)
a
Note that incubations of all P450s were performed as described
a
in Experimental Procedures. The amount of 4-hydroxy-236HCB
from L363V was 2.76 nmol h-1 100 pmol of P450-1
.
236HCB production, respectively. P450 2B11 mutants
V114I and D290I retained ∼50% of the level of wild-type
4,5-dihydroxy-236HCB production. Interestingly, the
substitution of Leu-363 with Val dramatically changed
the profile of 236HCB metabolism. In addition to a 1.5-
fold increase in the level of 4,5-dihydroxy-236HCB for-
mation, the mutant formed the novel metabolite 4-hy-
droxy-236HCB at a relatively high rate. The 4-hydroxy-
236HCB yield for the mutant was 2.76 nmol h-1 100 pmol
F igu r e 3. Profile of metabolites of 236HCB produced by P450
2B1 mutants.
236HCB, and M-3 was confirmed as 4-hydroxy-236HCB
(Figure 2).
Mon oh yd r oxy-236HCB Meta bolism by P 450 2B1
a n d 2B11 a n d 2B11 Mu ta n t L363V. As described
above, M-1 was identified as a dihydroxy-236HCB and
could be a secondary metabolite of M-2. To examine this
possibility, P450 2B1 wild type was used in assays of
monohydroxy-236HCB metabolism. Incubation of P450
2B1 with 5-hydroxy-236HCB or with M-2 produced a
single metabolite, which had an HPLC retention time
identical to that of 4,5-dihydroxy-236HCB (not shown).
Identification of the product as 4,5-dihydroxy-236HCB
was also confirmed by comparing the retention time on
GC of the trimethylsilyl derivative and fragmentation
pattern of mass spectra with those of the corresponding
standard (not shown). Interestingly, 4,5-dihydroxy-
236HCB was also formed by P450 2B1 from 4-hydroxy-
236HCB. Using 4-hydroxy-236HCB as the substrate,
2B11 wild type and 2B11 mutant L363V also produced
4,5-dihydroxy-236HCB, although the mutant exhibited
2-fold lower activity than the wild type (data not shown).
236HCB Meta bolism by P 450 2B1 a n d Its Mu -
ta n ts. The total amounts of 4,5-dihydroxy-236HCB and
5-hydroxy-236HCB produced by P450 2B1 wild type were
10.8 and 15.6 nmol h-1 100 pmol of P450-1, respectively.
All mutants yielded the same metabolites as the wild
type except I114V, which produced 4-hydroxy-236HCB
(0.3 nmol h-1 100 pmol of P450-1). Figure 3 shows the
236HCB metabolism profiles of P450 2B1 mutants. The
substitution of Ile-477 with Val had no significant effect
on 236HCB metabolism. The mutants I114V, F206L,
V363A, V363L, V367A, I477A, I477L, G478S, I480A, and
I480L exhibited product yields lower than that of the wild
type. The most striking decrease in activity was observed
with the L209A mutant, which had only 9 and 7% of the
wild-type 4,5-dihydroxy-236HCB and 5-hydroxy-236HCB
production, respectively. T302S exhibited the same amount
of 4,5-dihydroxy-236HCB yield, but only retained one-
third of the level of 5-hydroxy-236HCB formation as the
wild type. I480V produced twice as much dihydroxy
product as the wild type. Thus, I480V was the only P450
2B1 mutant that exhibited enhanced 236HCB metabo-
lism.
of P450-1
.
Discu ssion
In the study presented here, we have investigated the
metabolism of 236HCB by P450 2B1 and 2B11 wild type
and mutant enzymes. Two metabolites were produced by
all the samples and were identified as 4,5-dihydroxy-
236HCB and 5-hydroxy-236HCB. In addition, P450 2B11
mutant L363V and 2B1 mutant I114V formed the novel
metabolite 4-hydroxy-236HCB. 4,5-Dihydroxy-236HCB
was also obtained as a secondary metabolite of 5-hydroxy-
236HCB. The 5-hydroxy-236HCB may be the result of a
meta-para arene oxide intermediate which isomerized
to the 5-position (39, 40), and the 4,5-dihydroxy-236HCB
could be formed from 5-hydroxy-236HCB by a direct
hydroxylation mechanism for P450 2B1 wild type, 2B11
wild type, and most of their mutants. In the case of 2B11
L363V and 2B1 I114V, 4,5-dihydroxy-236HCB may be
produced from 5-hydroxy-236HCB and/or 4-hydroxy-
236HCB by a direct hydroxylation. In a study of
2,4,6,2′,4′,6′-HCB metabolism, Ariyoshi et al. (41) also
found that a dihydroxy PCB was produced from the
monohydroxy compounds by dog liver microsomes.
Identification of P450 2B mutants that exhibited
altered steroid or PCB metabolite profiles has provided
a powerful approach for identifying determinants of
substrate specificity. For example, P450 2B11 mutants
V107I, M199L/N200E/V204R, V234I, A292L, Q473R, and
I475S showed no differences from wild-type P450 2B11
in metabolite profiles with either androstenedione or
245HCB (20). Mutants V114I, D290I, L363V, and L480V
exhibited altered androstenedione metabolite profiles,
and three of them (except L480V) also displayed altered
profiles of 245HCB metabolites (20, 21). In this study,
the residues at positions 114, 206, 209, 302, 363, 367,
477, 478, and 480 in 2B1 and residues at positions 114,
290, and 363 in 2B11, which play the important role in
determining regio- and stereoselectivity of androstene-
dione metabolism (19, 20, 22), had a significant effect on
236HCB hydroxylase activity. Most mutants exhibited
lower hydroxylation activities than the wild type, al-
though the substitution of Leu-480 in 2B1 and Leu-363
in 2B11 with Val increased the level of 236HCB hydroxy-
236HCB Meta bolism by P 450 2B11 a n d Its Mu -
ta n ts. Table 1 shows the amount of 236HCB metabolites
produced by P450 2B11 and mutants. P450 2B11 wild
type only displayed one-fifteenth and one-fiftieth of the
level of 2B1 4,5-dihydroxy-236HCB and 5-hydroxy-

698 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Waller et al.
animal species. For example, a marked species variation
exists in the metabolism of 245HCB, and it has been
shown that among dogs, monkeys, rats, and humans,
only dogs are capable of metabolizing this compound to
a significant degree (38, 42). In our previous study, the
three residues at positions 114, 290, and 363 in the dog
2B11 were mutated to the corresponding residues in 2B1.
For all the mutants, the level of hydroxylation of 245HCB
was decreased (20). However, 2B1 reciprocal mutants did
not exhibit any increase in 245HCB hydroxylase activity
(data not shown). Unlike 245HCB, 236HCB can be
metabolized by both P450 2B1 and 2B11, although 2B1
metabolizes 236HCB at a much higher rate than 2B11.
Replacement of residues in P450 2B1 with the corre-
sponding residues of 2B11 (Table 2) decreased the
236HCB hydroxylase activities (I114V, V363L, and I480L)
or did not affect 236HCB metabolism (I477V), and
replacement of Leu-363 in 2B11 with the corresponding
residue Val of 2B1 significantly increased 236HCB hy-
droxylase activities. In contrast, the Val-114 f Ile and
Asp-290 f Ile substitution in 2B11 decreased the rate
of 236HCB metabolism. The lower activity of 2B11 D290I
may be due to the decreased stability of the mutated
protein (43).
F igu r e 4. Androstenedione and 236HCB hydroxylase activities
of P450 2B1 mutants.
Ta ble 2. Am in o Acid Sequ en ces of Cytoch r om e P 450 2B1
a n d 2B11 a t 10 P osition s
P450 114 206 209 290 302 363 367 477 478 480
2B1
2B11
I
V
F
F
L
I
I
D
T
T
V
L
V
V
I
V
G
G
I
L
Ta ble 3. Effect of Mu ta tion s in P 450 2B11 on Activity
w ith Va r iou s Su bstr a tesa
The hydroxylated metabolites of 236HCB in humans
have been previously idenfitied as 4- and 5-hydroxy-
236HCB (38). These assignments were based on the
characteristic fragmentation of methyl ether derivatives
of hydroxylated PCB metabolites (39, 40), and were not
supported by comparisons with authentic standards.
Studies with m- and p-methoxybiphenyl with one to five
chlorines on the biphenyl ring have shown that the two
compounds gave characteristic fragmentations. p-Meth-
oxy groups gave fragments corresponding to [M - CH3]
+, which has been explained by the formation of a quinoid
ion (39, 40). However, methylated 4-hydroxy-236HCB
had no fragment ion peak at [M - CH3] +, whereas the
methylated derivative of 5-hydroxy-236HCB afforded the
[M - CH3]+ fragment ion peak in our study. The same
phenomenon was also observed in several synthesized
compounds. For example, the mass spectra of 4-methoxy
groups in 2,3,6,2′,4′,6′-HCB, 2,3,6,2′,4′,5′-HCB, and
2,6,2′,4′,6′-pentachlorobiphenyl did not exhibit a fragment
ion at [M - CH3] +, whereas in the mass spectra of
3-methoxy-2,4,6,2′,4′,6′-HCB, the [M - CH3] + peak had
a relatively high intensity of 55% (44, 45). Thus, the
quinoid ion may be less stable in the case of 4-methoxy-
236HCB than 5-methoxy-236HCB. It appears that the
characteristic fragmentation differences in the mass
spectra of the m- and p-methoxy compounds are less
pronounced when the total number of chlorine atoms is
high. However, with the availability of authentic stan-
dards, as in this study, the two isomers can easily be
differentiated.
% of 2B11 wild-type activity
substrate or metabolite
V114I
D290I
L363V
androstenedione
16R-OH
16â-OH
245HCB
o-OH
m-OH
4
14
2
11
450
29
6
7
7
108
29
24
a
The data were taken from ref 20.
lation. 2B11 L363V produced 4-hydroxy-236HCB as its
major metabolite. This may be the result of a meta-para
arene oxide intermediate which mainly isomerized to the
4-position in the case of the mutant, and a low level of
further conversion of 4-hydroxy-236HCB to the dihydroxy
product.
Explanations for the differential effects of the muta-
tions observed with the various substrates (Figure 4 and
Table 3) are likely to lie in the unique orientations
adopted in the active site. For example, the replacement
of Leu-209 in 2B1 with Ala significantly decreased
236HCB hydroxylase activity to about 5% of the wild-
type activity, but the mutant still retained ∼70% of the
wild-type androstenedione hydroxylase activity. In con-
trast, substitution of Ile-480 in 2B1 with Val increased
236HCB and decreased androstenedione hydroxylase
activities (Figure 4). With 2B11, ortho hydroxylation of
245HCB is more sensitive than meta hydroxylation of
245HCB and 236HCB to the Val-114 f Ile substitution
(Tables 1 and 3). For the mutations at positions 290 and
363 in 2B11, 245HCB o- and m-hydroxylase activities
were decreased. In contrast, the 236HCB m-hydroxylase
activity was increased for L363V and unaltered for D290I
(Tables 1 and 3). Explanations for these observations may
come from modeling of the P450 together with docking
of the respective substrates, as has been done for P450
2B1 with androstenedione (19).
Ack n ow led gm en t. This work was supported by
Grant ES03619 and Core Center Grant ES06694 (Uni-
versity of Arizona) from the National Institutes of Health.
Su p p or tin g In for m a tion Ava ila ble: Characterization
details of compounds 9-12, 14-20, 23-30, 32-36, 38, and 39.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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