Hypersensitive radical probe studies of chloroperoxidase-catalyzed hydroxylation reactions

Article abstract of DOI:10.1021/tx9800295

The oxidation of hypersensitive radical probes by chloroperoxidase from Caldariomyces fumago (CPO) was studied in an attempt to 'time' a putative radical intermediate. Oxidation of (trans-2-phenylcyclopropyl)methane, previously studied by Zaks and Dodds [Zaks, A., and Dodds, D. R. (1995) J. Am. Chem. Soc. 115, 10419-10424] was reinvestigated. Unrearranged oxidation products were found as previously reported, and control experiments demonstrated that the cyclic alcohol from oxidation at the cyclopropylcarbinyl position, while subject to further oxidation, survives CPO oxidation as detectable species. However, in contrast to the report by Zaks and Dodds, the rearranged alcohol product expected from ring opening of a cyclopropylcarbinyl radical intermediate was shown to be unstable toward the enzyme oxidation reaction. Because of this instability, two new hypersensitive radical probes, (trans-2-phenylcyclopropyl)ethane and 2- (trans-2-phenylcyclopropyl)propane, and their potential cyclic and acyclic products from oxidation at the cyclopropylcarbinyl position were synthesized and tested. Oxidation of both of these probes at the cyclopropylcarbinyl position by CPO gave unrearranged alcohol products only, but control experiments again demonstrated that the rearranged alcohol products were unstable toward CPO oxidation conditions. From the combination of the probe and control studies, the lifetime of a putative radical intermediate must be less than 3 ps. Whereas the results are consistent with an insertion mechanism for production of alcohol product, they do not exclude a very short-lived intermediate.

Full text of DOI:10.1021/tx9800295

816
Chem. Res. Toxicol. 1998, 11, 816-823
Hyp er sen sitive Ra d ica l P r obe Stu d ies of
Ch lor op er oxid a se-Ca ta lyzed Hyd r oxyla tion Rea ction s
Patrick H. Toy,† Martin Newcomb,*,† and Lowell P. Hager‡
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, and
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
Received February 19, 1998
The oxidation of hypersensitive radical probes by chloroperoxidase from Caldariomyces
fumago (CPO) was studied in an attempt to “time” a putative radical intermediate. Oxidation
of (trans-2-phenylcyclopropyl)methane, previously studied by Zaks and Dodds [Zaks, A., and
Dodds, D. R. (1995) J . Am. Chem. Soc. 115, 10419-10424] was reinvestigated. Unrearranged
oxidation products were found as previously reported, and control experiments demonstrated
that the cyclic alcohol from oxidation at the cyclopropylcarbinyl position, while subject to further
oxidation, survives CPO oxidation as detectable species. However, in contrast to the report
by Zaks and Dodds, the rearranged alcohol product expected from ring opening of a
cyclopropylcarbinyl radical intermediate was shown to be unstable toward the enzyme oxidation
reaction. Because of this instability, two new hypersensitive radical probes, (trans-2-
phenylcyclopropyl)ethane and 2-(trans-2-phenylcyclopropyl)propane, and their potential cyclic
and acyclic products from oxidation at the cyclopropylcarbinyl position were synthesized and
tested. Oxidation of both of these probes at the cyclopropylcarbinyl position by CPO gave
unrearranged alcohol products only, but control experiments again demonstrated that the
rearranged alcohol products were unstable toward CPO oxidation conditions. From the
combination of the probe and control studies, the lifetime of a putative radical intermediate
must be less than 3 ps. Whereas the results are consistent with an insertion mechanism for
production of alcohol product, they do not exclude a very short-lived intermediate.
The consensus mechanism for hydrocarbon hydroxyl-
ation by P450 enzymes that evolved over the past 2
decades involves hydrogen abstraction by an “iron-oxo”
intermediate followed by homolytic substitution of the
radical at OH bound to iron (oxygen rebound step) (3-
5). However, recent mechanistic studies of hydroxylation
of hypersensitive radical probes by the rat liver isozyme
P450 2B1 (CYP2B1) have indicated that the “radical”
lifetime is too short for an intermediate and that the
reaction proceeds by an insertion mechanism with a
competing cation-forming pathway (6-9).
In tr od u ction
Chloroperoxidase from Caldariomyces fumago (CPO)1
is unique among the known heme-containing peroxidase
enzymes in that a thiolate from a protein cysteine serves
as the fifth ligand to iron (1). Thus, the active site of
CPO is related in gross architecture to those of the
ubiquitous cytochrome P450 (P450) enzymes. CPO is
also related in function to the P450 enzymes in that
several of the oxidation reactions effected by CPO are
similar to those of the P450 enzymes. The major differ-
ence between CPO and P450 enzymes is the source of
oxygen in nature; CPO employs hydrogen peroxide,
whereas P450 enzymes employ molecular oxygen and an
ancillary reduction system that uses reducing equivalents
from NAD(P)H (2). Nevertheless, because the reduction
of molecular oxygen in the P450 enzymes gives the formal
equivalent of hydrogen peroxide and given the similarity
of the oxidation reactions effected by CPO and P450
enzymes, there is a good likelihood that a common
oxidation mechanism exists for these enzymes. Accord-
ingly, mechanistic studies of CPO oxidations could pro-
vide results that have a direct bearing on the mechanisms
of P450 oxidations.
Although P450-catalyzed hydroxylations of hydrocar-
bons and the mechanism of this process have been widely
studied, relatively few such studies have been performed
with CPO. The first involved the hydroxylation of
cyclohexene (10), and the others studied the hydroxyla-
tion of benzylic positions in substrates (11, 12). One
mechanistic study of CPO hydroxylation, reported in a
paper focusing mainly on the use of CPO for stereose-
lective olefin epoxidations, resulted in the conclusion that
this hydroxylation reaction involves an insertion process
which occurs without the formation of a discrete radical
intermediate (11). In that study, the radical probe 1 was
hydroxylated to give cyclic alcohol 2, aldehyde 3, and acid
4 as the only products (eq 1). The authors reported that
no rearranged alcohol product 6, which would be pro-
duced from ring opening of the intermediate radical 5
(eq 2), was observed at a detection limit of at least 1 part
in 100 and that compound 6 was stable to the reaction
conditions.
* Address correspondence to: Prof. Martin Newcomb. Tel: (313)-
577-2782. Fax: (313)-577-8822. E-mail: men@chem.wayne.edu.
† Wayne State University.
‡ University of Illinois.
1
Abbreviations: CPO, chloroperoxidase; P450, cytochrome P450;
MMO, methane monooxygenase; fs, femtosecond; HRMS, high-resolu-
tion mass spectrometry; ether, diethyl ether; LAH, lithium aluminum
hydride; LHMDS, lithium bis(trimethylsilyl)amide.
S0893-228x(98)00029-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/19/1998

Probe Studies of Chloroperoxidase
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 817
ide. All substrates subjected to CPO oxidation were greater
than 98% pure as determined by GC analysis.
An a lytica l Meth od s. NMR spectra2 were acquired on a
Varian Gemini 300 spectrometer. Quantitative GC analyses
were performed with a Varian 3400 chromatograph equipped
with a flame-ionization detector on a 15-m × 0.54-mm bonded
phase Carbowax column (Alltech). GC/MS analyses were
performed with a Hewlett-Packard model 5890 chromatograph
interfaced to a Hewlett-Packard model 5971 mass selective
detector on a 30-m × 0.25-mm capillary bonded phase Carbowax
column (Alltech). HRMS (high-resolution mass spectrometry)
analyses were performed by the Central Instrumentation Facil-
ity at Wayne State University (Detroit, MI). Melting points
were determined using a Unimelt capillary melting point
apparatus (Thomas-Hoover) and are uncorrected. Radial chro-
matography was performed on a Chromatotron model 7294T
(Harrison Research Corp.) using plates coated with TLC grade
silica gel with gypsum binder and fluorescent indicator (Merck).
En zym a tic Oxid a tion Rea ction s. The enzymatic oxida-
tions of the substrates were performed as previously reported
with the exception that the substrate was added without the
aid of acetone (18). In a typical hydroxylation reaction, 1 mg
(25 nmol) of CPO was dissolved in 4.5 mL of 100 mM citrate
buffer (pH 5.0) in a 5-mL flask fitted with a magnetic stirrer
The mechanistic study of CPO hydroxylation of probe
1 provided interesting results which are, nonetheless, not
consistent with the kinetics of ring opening of radical 5.
The rate constant for rearrangement of this radical is so
great (kr ) 3 × 1011 s-1 at ambient temperature) (13) that
some rearranged product should have been observed even
if 5 was only a component of the transition-state struc-
ture; this point is developed in the discussion below. In
light of previous results using probe 1 in studies of the
mechanisms of hydroxylation by P450 enzymes (14, 15)
and by methane monooxygenase (MMO) systems (16), we
have reinvestigated the CPO hydroxylation of 1 in order
to resolve this apparent dichotomy and to attempt to
uncover mechanistic similarities between CPO hydroxy-
lation reactions and those of P450 and MMO enzymes.
We have also studied the CPO hydroxylation of two other
hypersensitive radical probes, 7 and 8 (eqs 3 and 4),
which have not previously been used to study enzymatic
processes. The selection of probe 7, previously used in a
study of Gif oxidations (17), and probe 8 was based on
the premise that oxidation at their cyclopropylcarbinyl
positions would produce the analogous cyclic products (9
and 10, and 12, respectively) as well as their ring-opened
alcohols (11 and 13, respectively). We report that,
whereas all three probes are oxidized by CPO, the
rearranged alcohols 6, 11, and 13 are not stable to the
reaction conditions. A very short maximum lifetime for
a putative radical intermediate in CPO hydroxylation can
be calculated from the results, but unequivocal conclu-
sions regarding an insertion mechanism for hydroxyla-
tion cannot be made.
and kept in
a constant-temperature bath at 25 °C. The
substrate (50 µmol) was added either neat or as a buffer solution,
and this mixture was allowed to equilibrate for 10 min. The
reaction was initiated by the addition of 0.75% hydrogen
peroxide at a rate of 0.1 mL/h via a syringe pump. After a total
of 0.5 mL of hydrogen peroxide solution had been added, the
reaction mixture was extracted with CH2Cl2 (3 × 2 mL).
The combined organic phase was dried over anhydrous
MgSO4 and filtered through a plug of glass wool. An internal
standard was added, and the solution was concentrated under
a slow stream of nitrogen at room temperature to a final volume
of approximately 0.2 mL. Quantitation of the product ratios
was performed by GC, and product identities were determined
by GC/MS by comparison with authentic samples using selected
ion monitoring. Carboxylic acid 4 was analyzed as its methyl
ester; thus, samples were treated with an ethereal solution of
CH2N2 and allowed to stand in a fume hood overnight before
analysis.
Control experiments to determine the stability of the rear-
ranged products were performed as above with samples of the
parent probe doped with small amounts of the putative alcohol
product.
Meth yl (tr a n s-2-P h en ylcyclop r op yl)a ceta te (14). To a
solution of (trans-2-phenylcyclopropyl)acetic acid (13) (6.00 g,
34.0 mmol) in CH2Cl2 (100 mL) was added a solution of CH2N2
in ether (19) until a slight yellow color persisted. The solution
was stirred for 16 h and then was concentrated in vacuo. The
crude product was chromatographed on silica gel (10% ethyl
acetate in hexanes) to afford 14 (6.02 g, 31.6 mmol, 93%) as a
clear, colorless oil. 1H NMR (CDCl3): δ 0.84 (1H, dt, J 1 ) 9.3
Hz, J 2 ) 5.4 Hz), 1.02 (1H, dt, J 1 ) 9.0 Hz, J 2 ) 5.4 Hz), 1.34-
1.45 (1H, m), 1.77 (1H, dt, J 1 ) 9.3 Hz, J 2 ) 5.1 Hz), 2.31-2.51
(2H, m), 3.70 (3H, s), 7.07-7.30 (5H, m).
2-(tr a n s-2-P h en ylcyclop r op yl)eth a n ol (15). To a solution
of 14 (0.82 g, 4.31 mmol) in THF (25 mL) at 0 °C was added
lithium aluminum hydride (LAH) (0.40 g, 10.5 mmol). The
mixture was stirred under a nitrogen atmosphere for 2 h, and
the reaction was quenched by the sequential addition of water
(0.4 mL), 15% aqueous NaOH solution (0.4 mL), and water (1.2
mL). The resulting suspension was stirred at room temperature
for an additional hour and then filtered and concentrated in
vacuo. The crude product was purified by radial chromatogra-
phy (40% ethyl acetate in hexanes) to afford 15 (0.56 g, 3.45
mmol, 80%) as a clear, colorless oil. 1H NMR (CDCl3): δ 0.83
Exp er im en ta l P r oced u r es
Ca u tion : All synthetic reactions should be performed in
properly ventilated hoods using published procedures for safe
handling.
Gen er a l Meth od s a n d Ma ter ia ls. CPO from C. fumago
was provided by Chirazyme. Commercially available starting
materials and reagents were purchased from either Sigma or
Aldrich Chemical Co. and were used as received. All moisture-
sensitive reactions were carried out in flame-dried glassware
under a nitrogen atmosphere. Tetrahydrofuran (THF) and
diethyl ether (ether) were distilled under a nitrogen atmosphere
over sodium and benzophenone ketyl. Methylene chloride was
distilled under a nitrogen atmosphere over phosphorus pentox-
2
Compounds that were previously reported in the literature but
were prepared by different methods and compounds which were
isolated as mixtures of diastereomers were not necessarily character-
ized by 13C NMR or HRMS.

818 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Toy et al.
(1H, ddd, J 1 ) 8.7 Hz, J 2 ) 5.4 Hz, J 3 ) 4.5 Hz), 0.94 (1H, dt,
J 1 ) 8.4 Hz, J 2 ) 4.8 Hz), 1.04-1.15 (1H, m), 1.46 (1H, bs),
1.58-1.75 (3H, m), 3.77 (2H, dd, J 1 ) 10.5 Hz, J 2 ) 6.3 Hz),
7.03-7.07 (2H, m), 7.14 (1H, tt, J 1 ) 6.9 Hz, J 2 ) 1.2 Hz), 7.22-
7.28 (2H, m). 13C NMR (CDCl3): δ 15.6, 20.2, 22.8, 37.3, 62.8,
125.4, 125.6 (2C), 128.3 (2C), 143.4. HRMS: calcd for C11H14O,
162.1045; found, 162.1045.
layer was extracted with ether (3 × 25 mL). The combined
organic phase was washed with brine (50 mL), dried over
MgSO4, filtered, and concentrated in vacuo at 0 °C. The crude
product was purified by radial chromatography (pentane) to
afford 8 (0.19 g, 1.19 mmol, 60%) as a clear, colorless oil. 1H
NMR (CDCl3): δ 0.76-0.88 (3H, m), 1.01-1.16 (1H, m), 1.02
(3H, d, J ) 4.5 Hz), 1.03 (3H, d, J ) 3.9 Hz), 1.66 (1H, dt, J 1
)
8.7 Hz, J 2 ) 4.8 Hz), 7.05-7.16 (3H, m), 7.22-7.27 (2H, m). 13
C
(tr a n s-2-P h en ylcyclop r op yl)eth a n e (7). A solution of 15
(0.41 g, 2.53 mmol) in THF (25 mL) under a nitrogen atmo-
sphere was cooled to -30 °C. To this were added sequentially
via syringe triethylamine (0.75 mL, 5.38 mmol) and methane-
sulfonyl chloride (0.25 mL, 3.23 mmol). The mixture was stirred
at -30 °C for 30 min and then cooled to -78 °C. A solution of
LiBEt3H (1.0 M in THF, 10.0 mL, 10.0 mmol) was added via
syringe. The mixture was allowed to warm slowly to room
temperature and was stirred for 16 h. The reaction was
quenched by the addition of 30% H2O2 solution (5 mL) and 15%
aqueous NaOH solution (5 mL). The resulting mixture was
heated at reflux for 1 h and then cooled to room temperature.
The organic layer was separated, and the aqueous layer was
extracted with ether (3 × 25 mL). The combined organic phase
was washed with brine (50 mL), dried over MgSO4, filtered, and
concentrated in vacuo at 0 °C. The crude product was purified
by radial chromatography (pentane) to afford 7 (0.24 g, 1.64
mmol, 65%) as a clear, colorless oil. 1H NMR (CDCl3): δ 0.85
(1H, ddd, J 1 ) 8.7 Hz, J 2 ) 5.7 Hz, J 3 ) 4.8 Hz), 0.96 (1H, dt,
J 1 ) 8.4 Hz, J 2 ) 5.1 Hz), 1.06-1.13 (1H, m), 1.17 (3H, t, J )
7.5 Hz), 1.44-1.55 (1H, m), 1.49 (2H, q, J ) 7.2 Hz), 1.71 (1H,
dt, J 1 ) 8.7 Hz, J 2 ) 4.5 Hz), 7.13-7.24 (3H, m), 7.30-7.36 (2H,
m). 13C NMR (CDCl3): δ 13.6, 16.1, 23.1, 25.6, 27.6, 125.2, 125.7
(2C), 128.3 (2C), 144.2. HRMS: calcd for C11H14, 146.1096;
found, 146.1099.
NMR (CDCl3): δ 15.1, 21.8, 22.1, 22.4, 31.7, 33.5, 125.1, 125.7
(2C), 128.1 (2C), 144.1. HRMS: calcd for C12H16, 160.1252;
found, 160.1255.
1-(tr a n s-2-P h en ylcyclop r op yl)eth a n on e (9). A solution
of N-methoxy-N-methyl-trans-2-phenylcyclopropanecarboxam-
ide (20) (6.10 g, 29.7 mmol) in ether (250 mL) under a nitrogen
atmosphere was cooled to 0 °C. To this was added a solution of
methylmagnesium bromide (3.0 M in THF, 10.0 mL, 30.0 mmol).
After 6 h, the reaction was quenched by pouring it into saturated
aqueous NH4Cl solution (250 mL), and then the mixture was
extracted with ether (3 × 150 mL). The combined organic phase
was washed with water (200 mL) and brine (200 mL), dried over
MgSO4, filtered, and concentrated in vacuo. The crude product
was chromatographed on silica gel (15% ethyl acetate in
hexanes) to afford 9 (4.08 g, 25.5 mmol, 86%) as a clear, colorless
oil. 1H NMR (CDCl3): δ 1.38 (1H, ddd, J 1 ) 8.4 Hz, J 2 ) 6.9
Hz, J 3 ) 4.5 Hz), 1.68 (1H, ddd, J 1 ) 9.3 Hz, J 2 ) 5.4 Hz, J 3
)
4.2 Hz), 2.22 (1H, ddd, J 1 ) 8.4 Hz, J 2 ) 5.4 Hz, J 3 ) 4.2 Hz),
2.31 (3H, s), 2.52 (1H, ddd, J 1 ) 9.0 Hz, J 2 ) 6.6 Hz, J 3 ) 4.2
Hz), 7.07-7.11 (2H, m), 7.18-7.32 (3H, m). 13C NMR (CDCl3):
δ 19.1, 28.9, 30.7, 32.8, 126.0 (2C), 126.5, 128.5 (2C), 140.3,
206.5. MS: calcd for C11H12O, 160; (M)+ found at m/z ) 160.
1-(tr a n s-2-P h en ylcyclop r op yl)eth a n ol (10). To a solution
of 9 (1.02 g, 6.37 mmol) in THF (30 mL) at 0 °C was added LAH
(0.25 g, 6.59 mmol). The mixture was stirred under a nitrogen
atmosphere for 2 h, and the reaction was quenched by the
sequential addition of water (0.25 mL), 15% aqueous NaOH
solution (0.25 mL), and water (0.75 mL). The resulting suspen-
sion was stirred at room temperature for 1 h and then filtered.
The filtrate was concentrated in vacuo, and the resulting crude
product was purified by radial chromatography (20% ethyl
acetate in hexanes) to afford 10 (0.98 g, 6.04 mmol, 95%) as a
clear, colorless oil. GC analysis showed this to be a 2:1 mixture
of diastereomers. 1H NMR (CDCl3): δ 0.87-1.03 (2H, m), 1.24-
12.31 (1H, m), 1.32 and 1.34 (3H, d, J ) 6.0 Hz), 1.57 and 1.59
(1H, bs), 1.81 and 1.90 (1H, dt, J 1 ) 9.3 Hz, J 2 ) 5.1 Hz), 3.34-
3.44 (1H, m), 7.05-7.09 (2H, m), 7.12-7.19 (1H, m), 7.23-7.29
(2H, m). MS: calcd for C11H14O, 162; (M)+ found at m/z ) 162
for each isomer.
2-(tr a n s-2-P h en ylcyclop r op yl)p r op a n e (8). A solution of
14 (1.30 g, 6.83 mmol) in THF (50 mL) under a nitrogen
atmosphere was cooled to -78 °C. To this was added a solution
of lithium bis(trimethylsilyl)amide (LHMDS) (1.0 M in THF, 7.0
mL, 7.00 mmol). After 15 min, iodomethane (1.3 mL, 20.2
mmol) was added. The mixture was allowed to warm gradually
to room temperature, and after 16 h, the reaction mixture was
poured into saturated, aqueous NH4Cl solution (100 mL) and
extracted with ether (3 × 50 mL). The combined organic phase
was washed with water (75 mL) and brine (75 mL), dried over
MgSO4, filtered, and concentrated in vacuo. The crude product
was chromatographed on silica gel (10% ethyl acetate in
hexanes) to afford a mixture of diastereomers of methyl 2-(trans-
2-phenylcyclopropyl)propionate (0.58 g, 2.84 mmol, 42%) as a
clear, colorless oil.
To a solution of the above mixture of esters (0.58 g, 2.84
mmol) in THF (50 mL) at 0 °C was added LAH (0.20 g, 5.27
mmol). The mixture was stirred under a nitrogen atmosphere
for 2 h, and the reaction was quenched by the sequential
addition of water (0.2 mL), 15% aqueous NaOH solution (0.2
mL), and water (0.6 mL). The resulting suspension was stirred
at room temperature for an additional hour and then filtered.
Concentration of the filtrate in vacuo gave crude product which
was purified by radial chromatography (40% ethyl acetate in
hexanes) to afford 2-(trans-2-phenylcyclopropyl)-1-propanol (0.36
g, 2.04 mmol, 72%) as a clear, colorless oil. GC analysis showed
this to be a 3:1 mixture of diastereomers. MS: calcd for
C12H16O, 176; (M)+ found at m/z ) 176 for each isomer.
A solution of the above alcohols (0.35 g, 1.99 mmol) in THF
(15 mL) under a nitrogen atmosphere was cooled to -30 °C. To
this was added sequentially via syringe triethylamine (0.50 mL,
3.59 mmol) and methanesulfonyl chloride (0.18 mL, 2.33 mmol).
The mixture was stirred at -30 °C for 30 min and then cooled
to -78 °C. A solution of LiBEt3H (1.0 M in THF, 10.0 mL, 10.0
mmol) was added via syringe. The mixture was allowed to
warm slowly to room temperature and was stirred for 16 h. The
reaction was quenched by the addition of 30% H2O2 solution (5
mL) and 15% aqueous NaOH solution (5 mL). The resulting
mixture was heated at reflux for 1 h and then cooled to room
temperature. The organic layer was separated, and the aqueous
(E)- a n d (Z)-1-P h en yl-3-p en ten -1-ol (11). A solution of 10
(0.43 g, 2.65 mmol) in THF (25 mL) under a nitrogen atmo-
sphere was cooled to -30 °C. To this were added sequentially
via syringe triethylamine (0.75 mL, 5.38 mmol) and methane-
sulfonyl chloride (0.25 mL, 3.23 mmol). After 1 h, water (15
mL) was added, and the reaction mixture was heated at reflux
for 2 h. After cooling to room temperature, the reaction mixture
was extracted with ether (3 × 25 mL). The combined organic
phase was washed with saturated, aqueous NaHCO3 solution
(50 mL) and brine (50 mL), dried over MgSO4, and filtered.
Concentration of the filtrate in vacuo gave crude product which
was purified by radial chromatography (15% ethyl acetate in
hexanes) to afford 11 (0.28 g, 1.73 mmol, 65%) as a clear,
colorless oil. GC analysis showed this to be a 5:1 mixture of
isomers. 1H NMR (CDCl3): δ 1.59-1.71 (3H, m), 2.01 (1H, bs),
2.34-2.63 (2H, m), 4.68 and 4.72 (1H, dd, J 1 ) 7.8 Hz, J 2 ) 4.8
Hz), 5.37-5.49 (1H, m), 5.55-5.68 (1H, m), 7.23-7.37 (5H, m).
MS: calcd for C11H14O, 162; (M)+ found at m/z ) 162 for each
isomer.
2-(tr a n s-2-P h en ylcyclop r op yl)-2-p r op a n ol (12). A solu-
tion of 9 (1.71 g, 10.7 mmol) in ether (50 mL) under a nitrogen
atmosphere was cooled to 0 °C. To this was added a solution of
methylmagnesium bromide (3.0 M in THF, 4.0 mL, 12.0 mmol).
After 6 h, the reaction was quenched by pouring it into
saturated, aqueous NH4Cl solution (100 mL), and the resulting

Probe Studies of Chloroperoxidase
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 819
Sch em e 1. Syn th esis of P r obe Su bstr a tes 7 a n d 8a
mixture was extracted with ether (3 × 75 mL). The combined
organic phase was washed with water (75 mL) and brine (75
mL), dried over MgSO4, and filtered. Concentration of the
filtrate in vacuo gave crude product that was chromatographed
on silica gel (20% ethyl acetate in hexanes) to afford 12 (1.49 g,
8.45 mmol, 79%) as a clear, colorless oil. 1H NMR (CDCl3): δ
0.85 (1H, dt, J 1 ) 8.7 Hz, J 2 ) 4.8 Hz), 1.01-1.07 (1H, m), 1.19
(1H, s), 1.23-1.30 (1H, m), 1.30 (6H, s), 1.94 (1H, dt, J 1 ) 8.7
Hz, J 2 ) 5.1 Hz), 7.07-7.18 (3H, m), 7.62 (2H, tt, J 1 ) 7.8 Hz,
J 2 ) 2.1 Hz). 13C NMR (CDCl3): δ 11.6, 19.2, 28.9, 29.1, 34.1,
69.4, 125.4, 126.0 (2C), 128.3 (2C), 143.2. MS: calcd for
C12H16O, 176; (M)+ found at m/z ) 176.
4-Meth yl-1-ph en yl-3-pen ten -1-ol (13). A solution of 2-phen-
yl-1,3-dithiane (2.95 g, 15.0 mmol) in THF (50 mL) under a
nitrogen atmosphere was cooled to -20 °C, and BuLi (2.5 M in
hexanes, 6.0 mL, 15.0 mmol) was added. After 1 h, the solution
was cooled to -78 °C, and 1-bromo-3-methyl-2-butene (2.6 mL,
22.5 mmol) was added. The mixture was allowed to warm
slowly to room temperature with stirring and was stirred for a
total of 16 h. The reaction was quenched by pouring the mixture
into saturated, aqueous NH4Cl solution (100 mL). The mixture
was extracted with ether (3 × 50 mL). The combined organic
phase was washed with brine (75 mL), dried over MgSO4, and
filtered. Concentration in vacuo gave the crude product that
was chromatographed on silica gel (10% ethyl acetate in
hexanes) to afford 2-(3-methyl-2-butenyl)-2-phenyl-1,3-dithiane
(3.41 g, 12.9 mmol, 86%) as a yellow oil.
a
(a) CH2N2, ether; (b) LAH, THF, 0 °C; (c) (i) MsCl, Et3N, THF,
-30 °C, (ii) LiEt3BH, -78 °C; (d) (i) LHMDS, THF, -78 °C, (ii)
MeI.
Sch em e 2. Syn th esis of P u ta tive Oxid a tion
P r od u cts 9-13a
To a suspension of CaCO3 (0.25 g, 2.49 mmol) and Hg-
(ClO4)2‚xH2O (0.87 g, 2.18 mmol) in methanol (15 mL) was added
a solution of the above dithiane (0.52 g, 1.97 mmol) in CH2Cl2
(5 mL). The mixture was stirred at room temperature for 10
min and then filtered through Celite. Solvent was removed in
vacuo, and the crude product was chromatographed on silica
gel (10% ethyl acetate in hexanes) to afford 4-methyl-1-phenyl-
3-penten-1-one (0.28 g, 1.62 mmol, 82%) as a clear, colorless oil.
A solution of the above ketone (0.26 g, 1.49 mmol) in THF
(25 mL) was cooled to 0 °C under a nitrogen atmosphere, and
LAH (0.10 g, 2.64 mmol) was added. The mixture was stirred
for 2 h, and the reaction was quenched by the sequential
addition of water (0.1 mL), 15% aqueous NaOH solution (0.1
mL), and water (0.3 mL). The resulting mixture was stirred
for 1 h and then filtered. Concentration of the filtrate in vacuo
gave crude product that was purified by radial chromatography
(10% ethyl acetate in hexanes) to afford 13 (0.25 g, 1.42 mmol,
95%) as a clear, colorless oil. 1H NMR (CDCl3): δ 1.61 (3H, s),
a
(a) MeMgBr, ether, 0 °C; (b) LAH, THF, 0 °C; (c) (i) MsCl,
Et3N, THF, -30 °C, (ii) water reflux; (d) (i) BuLi, THF, -20 °C,
(ii) prenyl bromide; (e) Hg(ClO4)2‚xH2O, CaCO3, MeOH, CH2Cl2.
to probe 8 by a four-reaction sequence. It was first
methylated at the R-position, to provide the proper carbon
framework, by conversion to its lithium enolate by
treatment with LHMDS and reaction of the enolate with
iodomethane. This sequence gave a mixture of propi-
onate diastereomers that were not separated but used
directly since reduction of the ester group to a methyl
group renders the cyclopropylcarbinyl position achiral.
LAH reduction of the mixture of propionates gave the
corresponding propanols as a 3:1 mixture of diastereo-
mers. Conversion of the mixture of propanols to probe 8
was performed by the same two-step sequence as em-
ployed with 7.
Synthesis of the putative oxidation products from 7 and
8 also utilized trans-2-phenylcyclopropanecarboxylic acid
as the starting material (Scheme 2). The Weinreb amide
of this acid (20) was the key intermediate for the
formation of the cyclic products expected from both 7 and
8. Treatment of this amide with methylmagnesium
bromide afforded ethanone 9 (25). Reduction of 9 with
LAH afforded a 2:1 mixture of alcohols 10 (26). Meth-
anesulfonation of 10 followed by hydrolysis afforded a 5:1
mixture of ring-opened alcohols 11 (27). The cyclic
hydroxylation product 12 (28) was prepared by methyl-
magnesium bromide addition to 9. The acyclic hydroxy-
lation product 13 proved to be rather difficult to synthe-
size. Attempts to prepare 13 by methods analogous to
1.73 (3H, s), 1.81 (1H, s), 2.37-2.55 (2H, m), 4.68 (1H, dd, J 1
)
7.8 Hz, J 2 ) 5.4 Hz), 5.14-5.21 (1H, m), 7.24-7.38 (5H, m).
MS: calcd for C12H16O, 176; (M)+ found at m/z ) 176.
Resu lts
Syn t h eses of P r ob es a n d P u t a t ive Oxid a t ion
P r od u cts. The syntheses of probe 1 and its possible
oxidation products have been described (15, 16). Com-
pounds 7 and 8 were reported previously (21, 22), but
the syntheses involved carbenoid additions to alkenes
and did not give products in high diastereomeric purity.
Therefore alternative syntheses, divergent from a com-
mon intermediate, were designed (Scheme 1) in which
high diastereomeric purity was available in the starting
material, trans-2-phenylcyclopropanecarboxylic acid. This
acid was homologated to the corresponding (trans-2-
phenylcyclopropyl)acetic acid by a literature method (13).
Conversion of the acetic acid derivative to its methyl ester
(14) (23) was achieved by treatment with diazomethane.
Ester 14 proved to be a versatile intermediate for the
synthesis of probes 7 and 8. Reduction of 14 with LAH
afforded alcohol 15 (24), which was reduced to hydrocar-
bon probe 7 by the sequence of methanesulfonation
followed by hydride reduction. Ester 14 was converted

820 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Ta ble 1. P r od u cts fr om CP O Oxid a tion s w ith 25 n m ol of En zym ea
Toy et al.
those used to prepare 11 proved futile, but it was
available from a three-step sequence starting from
2-phenyl-1,3-dithiane by modification of a literature
method (29). Alkylation of the dithiane with prenyl
bromide provided the proper carbon skeleton. Removal
of the dithioacetal proved to be difficult, but experiments
showed that mercuric perchlorate was the reagent of
choice for conversion to the corresponding ketone. This
ketone was reduced with LAH to afford 13.
CP O Oxid a tion Rea ction s. Probes 1, 7, and 8 were
oxidized by CPO (Table 1). A dramatic steric effect in
the hydroxylation reactions was apparent with the yields
of oxidized products dropping precipitously as the steric
bulk of the probe increased. This is unlike the case with
the isozyme P450 2B1 where the same series of probes
was found to react with only a minor decrease in yield
with increase in steric bulk.3 Nevertheless, hydroxyla-
tion products were identified from each of the probes. In
the case of probe 1, alcohol 2, aldehyde 3, and acid 4 were
produced, but no ring-opened product 6 was observed.
These results essentially reproduce those reported by
Zaks and Dodds (11).
Reactions of probe 7 gave alcohol 10 (2.5:1 mixture of
diastereomers) as the only identifiable products, and
reactions of probe 8 gave only alcohol 12. Due to the low
yields of hydroxylation products and the presence of small
amounts of unidentified volatile components from the
enzyme reactions, the quantitative sensitivity of the
analyses was compromised. However, the identities of
products 10 and 12 were confirmed by GC/MS compari-
son to the respective authentic samples. In the case of
probe 7, the absence of ketone 9 and ring-opened alcohol
11 was firmly established by the absence of detectable
GC peaks corresponding to those of the authentic samples.
In a similar manner, no detectable GC peak correspond-
ing to that of authentic ring-opened alcohol 13 was
present from oxidation studies with probe 8.
Zaks and Dodds reported that ring-opened alcohol 6
from hydroxylation of probe 1 was stable to the enzyme
oxidation conditions (11). We found in control experi-
ments, however, that alcohol 6 was not stable (Table 1).
CPO oxidations of small amounts of authentic 6 returned
none of the alcohol. These experiments were designed
such that the amount of alcohol 6 employed was ap-
proximately equal to the amount of ring-opened hydroxy-
lation product expected if the ratio of unrearranged to
rearranged products was similar to that observed in P450
hydroxylations of probe 1 (14, 15); specifically, the
amounts of 6 were 0.3% and 1% of the amount of probe
1. Further, when the CPO oxidation of probe 1 was
repeated with a sample intentionally contaminated with
0.1% of alcohol 6, none of the acyclic alcohol was observed
by GC analysis. CPO oxidation of a sample of probe 1
containing 1.3% of alcohol 6 did yield a product mixture
containing some 6, but quantitative GC analysis showed
that only about 5% of the initial amount of 6 was
returned from this reaction. That the fully competent
CPO enzyme system was responsible for destruction of
alcohol 6 was demonstrated by control experiments in
which 6 was subjected to enzyme in buffer (without H2O2)
and to hydrogen peroxide in buffer (without enzyme); in
both cases, 6 was returned quantitatively. In the discus-
sion, we present a rationalization of the difference
between our control experiments with 6 and the result
reported by Zaks and Dodds (11).
Another control experiment was designed to determine
the stability of cyclic alcohol 2 toward CPO oxidation
conditions. The production of aldehyde 3 and acid 4 in
reactions of probe 1 clearly demonstrates that overoxi-
dation occurs, but we wished to determine whether a
significant portion of the initially formed alcohol 2 was
further processed to unknown products. A sample of
probe 1 doped with 0.2% of the perdeuteriophenyl ana-
logue of alcohol 2 was subjected to CPO oxidation, and
products 2 and 3 were analyzed by GC/MS. The total
yield of 2 and 3 was about 1% of the initial amount of 1,
3
Toy, P. H., Newcomb, M., and Hollenberg, P. F. Unpublished
results.

Probe Studies of Chloroperoxidase
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 821
consistent with the amounts of these products found from
probe 1. Alcohol 2 had a d0/d5 ratio of 2.5:1.0, and
aldehyde 3 had a d0/d5 ratio of 1.4:1.0. The internal
consistency of these values is excellent; because the
average concentration of 2-d0 will be about one-half of
the final concentration, the ratio of undeuterated to
deuterated 2 should be about twice as great as the ratio
for 3. The internally consistent ratios of d0- to d5-
products and the fact that the 2-d5 initially present was
largely recovered as 2 or 3 demonstrate that little of the
initially produced alcohol 2 was converted to unidentifi-
able products.
The observed instability of the ring-opened alcohol 6
was the impetus for attempting CPO oxidations of the
methyl- and dimethyl-substituted analogues of probe 1.
Our hope was that the ring-opened alcohol products from
probes 7 and 8 would be more stable than alcohol 6.
Unfortunately, control experiments demonstrated that
they were not. Thus, CPO oxidations of small amounts
of authentic alcohols 11 and 13 returned no detectable
amounts of these alcohols.
alcohols 2 and 6 increases for the same two P450 mutants
reported by Vaz et al. (30). In the CPO oxidation
reactions, a hydroperoxy-iron species will not be pro-
duced because the oxidizing equivalents are supplied by
hydrogen peroxide, and the absence of phenol 16 is
consistent with the premise that two electrophilic oxi-
dants are present in P450 and the latter, the iron-oxo
species, effects hydroxylations more efficiently than
epoxidations.
Because alcohol 6 was shown to be stable in oxidations
catalyzed by the P450 and MMO enzymes, the ratio of
unrearranged alcohol product 2 to rearranged alcohol
product 6 could be used with the known rate constant
for ring opening of cyclopropylcarbinyl radical 5 to
calculate a maximum lifetime for putative radical inter-
mediates in the hydroxylation reactions. The lifetimes
thus obtained for the MMO hydroxylase enzymes were
<100 fs and ca. 150 fs for M. capsulatus (Bath) and M.
trichosporium OB3b, respectively (16). For the P450
enzyme, the maximum lifetime for the putative radical
intermediate calculated from studies with probe 1 was
about 800 fs (14, 15), but subsequent mechanistic studies
of isozyme P450 2B1 employing other hypersensitive
radical probes have suggested that an interfering cationic
rearrangement occurs in P450 oxidations of probe 1 and
that the “radical” has a much shorter lifetime, only about
70 fs (6, 7).
Discu ssion
Probe 1 was previously used in mechanistic studies of
hydroxylation reactions by microsomal cytochrome P450
from livers of phenobarbital-treated rats and the purified
isozyme P450 2B1 (14, 15) and by the MMO hydroxylase
enzymes from Methylococcus capsulatus (Bath) and Me-
thylosinus trichosporium OB3b (16). In those studies,
probe 1 was oxidized to the cyclic alcohol 2, ring-opened
alcohol product 6, and phenol 16 (eq 5). Two notable
differences between CPO oxidation of 1 and oxidations
by the other enzymes studied are the overoxidation of
alcohol 2 by CPO, which has not been observed previ-
ously, and the absence of significant amounts of phenol
16 in the CPO oxidations.
The utility of the calibrated hypersensitive probes is
apparent from the above results. An intermediate cannot
have a lifetime of only 100 fs at ambient temperature
because the maximum translation of a second-row atom
in this period of time is only about 0.2 Å. Thus, the short
periods have been ascribed to the “lifetimes” of the
transition structures for insertion reactions that occur
in a nonsynchronous manner. Further, the small dis-
tance over which an atom can translate in such a period
has led to the conclusion that the carbon and oxygen
atoms were nearly within bonding distance of one an-
other at the instant of the insertion reaction.
Given the structural similarities of the active sites of
the P450 enzymes and CPO, it is reasonable to assume
that the mechanisms of the hydroxylation reactions
catalyzed by P450 and CPO are similar. Thus, the report
by Zaks and Dodds (11) that CPO oxidation of probe 1
gave only unrearranged oxidation products and that the
rearranged alcohol was stable was surprising. On the
basis of the results of P450 hydroxylations of 1 and other
hypersensitive probes, some rearranged alcohol product
6 should have been observed even if an insertion mech-
anism operated.
The absence of phenol 16 is noteworthy. This could
result from steric effects that prevent the appropriate
orientation of the substrate in the CPO active site, and
the dramatic reduction in the amounts of hydroxylation
observed for the series of probes 1, 7, and 8, which was
not observed in P450 2B1 hydroxylations of the same
series,3 suggests that steric effects are important. It is
possible, however, that the lack of production of phenol
16 is much more important from a mechanistic perspec-
tive. Vaz et al. recently reported that replacement of
active-site threonine by alanine in two P450 isozymes
results in an increase in epoxidation relative to allylic
hydroxylation in oxidations of alkenes (30). This result
indicates that two active electrophilic oxidants exist in
P450: a hydroperoxy-iron species that is long-lived when
threonine is not present in the active site and preferen-
tially epoxidizes and the ultimate oxidant, taken to be
iron-oxo, that preferentially hydroxylates. For probe 1,
phenol 16 is the “epoxidation” product, and we have found
in unpublished work4 that the ratio of phenol 16 to
Unfortunately, we found that alcohol 6 is not stable in
the presence of CPO when the amount of 6 present was
similar to the amounts formed as products in P450
oxidations of 1 (14, 15). Details of the control experi-
ments that led to the conclusion that 6 was stable were
not reported (11), but we assume that the reactions
involved relatively large amounts of 6. Our previous
studies have demonstrated that terminal alkenes and
alkynes alkylate the heme in CPO (31), and it is possible
that, in the control studies by Zaks and Dodds, the CPO
enzyme was destroyed rapidly and little of the test
substrate was consumed. In fact, if the amounts of 6 in
the control reactions were similar to the amounts of other
substrates used in that study, it is possible that the CPO
consumed less than 1% of the rearranged alcohol on the
4
Toy, P. H., Newcomb, M., Vaz, A. D. N., and Coon, M. J .
Unpublished results.

822 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Toy et al.
basis of our observation that about 0.6 µmol of 6 was
destroyed in the presence of 25 nmol of CPO.
lations by the P450 isozymes 2B4 and 2E1 give similar
results.4 In the case of MMO hydroxylations, “radical”
lifetimes of less than 200 fs have been implicated in
studies of two MMO hydroxylase enzymes with probe 1
(16), and importantly, the same short lifetimes were
found in oxidations of chiral ethane by the same enzymes
(32, 33). The conservative estimate that the lifetime of
a putative radical intermediate in CPO hydroxylation is
less than 3 ps provides no evidence against an insertion
pathway for hydroxylation.
The instability of the rearranged oxidation products
toward CPO oxidation conditions severely limits the
quantitative aspects of this probe study. We have
demonstrated that most of the unrearranged oxidation
product 2 survives in a detectable form (as 2, 3, or 4), so
the amounts of these products found in the CPO oxidation
reactions of probe 1 can be used as yields without
corrections. Further, we found that, when rearranged
alcohol 6 was initially added to the reaction in an amount
approximately equal to the amounts of unrearranged
products ultimately observed, a detectable amount of 6
was found at the end of the reaction. Therefore, it
appears to be safe to conclude that the amount of 6
produced in CPO oxidation of probe 1 was less than the
total amount of products 2-4. Accordingly, using the
rate constant for rearrangement of cyclopropylcarbinyl
radical 5 of 3 × 1011 s-1 at the reaction temperature (13),
one calculates that the lifetime of a putative radical
intermediate from probe 1 must be less than 3 ps. Due
to the small turnover values for probes 7 and 8, experi-
ments with the corresponding rearranged alcohol prod-
ucts similar to those performed with 1 were not deemed
productive, but the detection of unrearranged alcohol
products in both cases requires that “radical” lifetimes
were short.5
Ack n ow led gm en t. This research was supported in
part by grants from the National Institutes of Health
(GM-48722 to M.N., GM-07768 to L.P.H.).
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