A Substituted Hypersensitive Radical Probe for Enzyme-Catalyzed Hydroxylations: Synthesis of Racemic and Enantiomerically Enriched Forms and Application in a Cytochrome P450-Catalyzed Oxidation

Article abstract of DOI:10.1021/jo9712097

The syntheses of racemic and enantiomerically enriched trans-1-methyl-2-(4-(trifluoromethyl)phenyl)cyclopropane (3) and the possible oxidation products from enzyme-catalyzed hydroxylation of 3 at the methyl group are reported. The important intermediate in the production of 3 was the Weinreb amide of the 2-arylcyclopropanecarboxylic acid which could be prepared in diastereomerically pure form and which also served as an intermediate for production of the cyclic oxidation products of 3. Hydroxylation of 3 by the cytochrome P450 isozyme CYP2B1 gave cyclic and ring-opened products. The product ratios support an insertion mechanism for the enzyme-catalyzed hydroxylation reaction in which minor amounts of rearranged products are produced by radical fragmentation within the transition structure of the insertion and by a competing reaction involving a cationic species. Formation of cationic rearrangement products by a heterolytic fragmentation reaction of a first-formed protonated alcohol product is suggested on the basis of the apparent amounts of cationic products formed in the hydroxylation of 3. This pathway for cation production appears to require that the activated enzyme complex (equivalent to enzyme-substrate-H₂O₂) oxidizes substrate before water dissociates to give an iron-oxo species.

Full text of DOI:10.1021/jo9712097

9114
J . Org. Chem. 1997, 62, 9114-9122
A Su bstitu ted Hyp er sen sitive Ra d ica l P r obe for En zym e-Ca ta lyzed
Hyd r oxyla tion s: Syn th esis of Ra cem ic a n d En a n tiom er ica lly
En r ich ed F or m s a n d Ap p lica tion in a Cytoch r om e P 450-Ca ta lyzed
Oxid a tion
Patrick H. Toy,1a Bhavani Dhanabalasingam,1a Martin Newcomb,*,1a Imad H. Hanna,1b and
Paul F. Hollenberg*,1c
Departments of Chemistry and Pharmacology, Wayne State University, Detroit, Michigan 48202 and
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109
Received J uly 2, 1997X
The syntheses of racemic and enantiomerically enriched trans-1-methyl-2-(4-(trifluoromethyl)phen-
yl)cyclopropane (3) and the possible oxidation products from enzyme-catalyzed hydroxylation of 3
at the methyl group are reported. The important intermediate in the production of 3 was the
Weinreb amide of the 2-arylcyclopropanecarboxylic acid which could be prepared in diastereomeri-
cally pure form and which also served as an intermediate for production of the cyclic oxidation
products of 3. Hydroxylation of 3 by the cytochrome P450 isozyme CYP2B1 gave cyclic and ring-
opened products. The product ratios support an insertion mechanism for the enzyme-catalyzed
hydroxylation reaction in which minor amounts of rearranged products are produced by radical
fragmentation within the transition structure of the insertion and by a competing reaction involving
a cationic species. Formation of cationic rearrangement products by a heterolytic fragmentation
reaction of a first-formed protonated alcohol product is suggested on the basis of the apparent
amounts of cationic products formed in the hydroxylation of 3. This pathway for cation production
appears to require that the activated enzyme complex (equivalent to enzyme-substrate-H2O2)
oxidizes substrate before water dissociates to give an iron-oxo species.
Enzyme-catalyzed hydroxylations of unactivated C-H
positions at ambient temperatures represent one of the
most energetically difficult classes of reactions effected
by Nature. Such oxidations are commonly catalyzed by
cytochrome P450 enzymes which are broadly distributed
in animals, plants and bacteria.2,3 The active sites of
cytochrome P450 enzymes contain a heme with thiolate
from cysteine as the fifth ligand to iron. It is likely that
the substrate specificity and regio- and stereoselectivity
of cytochrome P450 enzymes results from the protein
portions of the enzymes and that a common mechanism
exists for numerous hydrocarbon hydroxylation reactions.
Considerable information concerning the timing of events
in cytochrome P450 hydroxylations is known, but the
mechanism of the hydroxylation step remains unresolved.
For the past two decades, the consensus mechanism
for cytochrome P450 hydroxylation involved pathway a
in Figure 1. Hydrogen atom abstraction from substrate
by a high valent iron-oxo intermediate produces an alkyl
radical intermediate (hydrogen abstraction step), and
subsequent homolytic displacement of OH from iron by
the radical gives alcohol product (oxygen rebound step).4-7
This mechanism, which displaced an oxygen insertion
process, was deduced mainly from the results of mecha-
F igu r e 1. The hydrogen abstraction-oxygen rebound mech-
anism (a) and insertion mechanism (b) for cytochrome P450
catalyzed hydroxylation.
nistic probe studies that implicated a radical intermedi-
ate by virtue of the formation of products expected from
radical rearrangements and is permissively supported by
the observation of kinetic isotope effects in hydroxylation
reactions. However, attempts to “time” the oxygen
rebound step with several fast, calibrated radical clocks
gave incongruous results.8-12 This suggested that an
unidentified process might be occurring in competition
with or as a part of the hydroxylation sequence. Because
a radical and a cationic intermediate would suffer the
same skeletal reorganization in each probe for which
rearranged products were found, the possibility of a side-
reaction involving a cation could not be excluded. We
X Abstract published in Advance ACS Abstracts, December 1, 1997.
(1) (a) Department of Chemistry, Wayne State University. (b)
Department of Pharmacology, Wayne State University. (c) Department
of Pharmacology, The University of Michigan.
(2) Cytochrome P450 Structure, Mechanism, and Biochemistry, 2nd
ed.; Ortiz de Montellano, P. R., Ed.; Plenum: New York, 1995.
(3) Guengerich, F. P. Am. Sci. 1993, 81, 440-447.
(4) (a) Groves, J . T.; Han, Y.-z. in ref 2; chapter 1. (b) Ortiz de
Montellano, P. R. in ref 2; chapter 8.
(8) Ortiz de Montellano, P. R.; Stearns, R. A. J . Am. Chem. Soc.
1987, 109, 3415-3420.
(9) Bowry, V. W.; Ingold, K. U. J . Am. Chem. Soc. 1991, 113, 5699-
5707.
(5) Woggon, W. D.; Fretz, H. In Advances in Detailed Reaction
Mechanisms; Coxon, J . M., Ed.; J AI: Greenwich, CT, 1992; Vol. 2; pp
111-147.
(10) Atkinson, J . K.; Ingold, K. U. Biochemistry 1993, 32, 9209-
9214.
(11) Atkinson, J . K.; Hollenberg, P. F.; Ingold, K. U.; J ohnson, C.
C.; Le Tadic, M.-H.; Newcomb, M.; Putt, D. A. Biochemistry 1994, 33,
10630-10637.
(12) Newcomb, M.; Le Tadic, M.-H.; Putt, D. A.; Hollenberg, P. F.
J . Am. Chem. Soc. 1995, 117, 3312-3313.
(6) Guengerich, F. P.; Macdonald, T. L. FASEB J . 1990, 4, 2453-
2459.
(7) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J . H. Chem. Rev.
(Washington, D.C.) 1996, 96, 2841-2887.
S0022-3263(97)01209-7 CCC: $14.00 © 1997 American Chemical Society

Radical Probe for Enzyme-Catalyzed Hydroxylations
J . Org. Chem., Vol. 62, No. 26, 1997 9115
Sch em e 1a
developed an ultrafast radical clock design that could be
used to differentiate between radical and cationic spe-
cies,13 and cytochrome P450 oxidation of one such probe
substrate, probe 1, demonstrated that both “radical” and
“cationic” rearrangements occurred in the hydroxylation
process.14 The qualitative observation of cation-derived
products explained the origin of the incongruous “radical
lifetimes” found from other radical probe/clocks. The
quantitative results indicated that the “radical lifetime”
in the cytochrome P450 hydroxylation was only 70
femtoseconds (fs), too short a lifetime for a true inter-
mediate.15 In other words, the “radical” could only exist
in the transition state ensemble for a nonsynchronous,
concerted hydroxylation (pathway b in Figure 1). Fur-
ther, the very short lifetime of the radical requires that
the carbon and oxygen atoms are situated nearly within
bonding distance of one another in the transition state,14
a conclusion that is supported by the regioselectivity
observed in enzyme-catalyzed hydroxylations of methyl-
cubane.16
The generality of the result found in the hydroxylation
of one probe by one cytochrome P450 isozyme must be
tested, but mechanistic studies of cytochrome P450
catalyzed hydroxylations are complicated for several
reasons. Many of these enzymes are highly selective in
regard to substrates and will not accept large mechanistic
probes. When the probe is accepted as a substrate,
oxidation often occurs at multiple sites. Further com-
plicating the situation, the less exclusive hepatic cyto-
chrome P450 enzymes from mammals are membrane-
bound, and the structures of these enzymes are not yet
known.
a
(a) Ph3PdCHC(O)N(OCH3)CH3, CH2Cl2 (95%); (b) (CH3)3SO+I-,
NaH, DMSO and then 4 (86%); (c) LiAlH4, THF, 0 °C (62%); (d)
i-Bu2AlH, CH2Cl2, -78 °C (79%); (e) t-BuOK, Et2O, H2O (90%); (f)
(i) MsCl, Et3N, THF, -30 °C, (ii) LiEt3BH, THF, -78 °C (79%);
(g) CH2N2, CH2Cl2 (94%).
Resu lts
Syn th esis of P r obe 3. Compound 3 was reported
previously from a carbenoid addition to the appropriate
substituted styrene.18 This preparation afforded a mix-
ture of diastereomers and was impractical for our use;
in addition to a diastereomer separation, alternative
routes to the possible oxidation products of the probe
would be required. Therefore, a synthesis of 3 was
designed which would yield the trans diastereomer and
some of its possible oxidation products (Scheme 1).
The commercially available 4-(trifluoromethyl)benzal-
dehyde was first converted to the Weinreb amide 4 by
Wittig olefination with N-methoxy-N-methyl-2-(triphen-
ylphosphoranylidene)acetamide.19 Compound 4 was pre-
pared so that the cyclopropanation protocol developed by
Rodriques20 could be employed. Accordingly, 4 was
treated with the ylide derived from trimethylsulfoxonium
iodide and sodium hydride to afford 5.
Amide 5 proved to be quite versatile in that it could
be converted into three of the possible oxidation products
of probe 3. Carbinol 6 was prepared by treatment of 5
with lithium aluminum hydride. Aldehyde 7 was readily
prepared by treatment of 5 with diisobutylaluminum
hydride (DIBALH) at low temperature; this compound
was found to be quite unstable and was prepared im-
mediately prior to use. Saponification of 5 by Gassman’s
“anhydrous hydroxide” procedure21 afforded acid 8. To
Phenyl groups in the cyclopropane probes provide the
driving force for the very fast radical ring opening
reactions (k > 1011 s-1 at ambient temperature) that are
necessary to time events in transition states, but the
phenyl groups in probes such as 2 are oxidized to
undesired phenols by cytochrome P450 enzymes10,11 and
by methane monooxygenase systems.17 We envisioned
substitution of the para position of the aromatic rings
with nonoxidizable groups as a means to avoid phenol
production and possibly to permit substituent effect
studies. We report here the syntheses of diastereomeri-
cally pure trans-1-methyl-2-(4-(trifluoromethyl)phenyl)-
cyclopropane (3) and the possible products from enzyme-
catalyzed oxidation of probe 3 at the methyl position,
resolution of 3 to high enantiomeric enrichment, and
cytochrome P450 catalyzed hydroxylations of racemic 3
and its enantiomers.
(13) Newcomb, M.; Chestney, D. L. J . Am. Chem. Soc. 1994, 116,
9753-9754.
(14) Newcomb, M.; Le Tadic-Biadatti, M.-H.; Chestney, D. L.;
Roberts, E. S.; Hollenberg, P. F. J . Am. Chem. Soc. 1995, 117, 12085-
12091.
(15) In their excellent review of heme-containing oxygenases, Sono
et al. (ref 7) state that the lifetimes we report contain calculation errors.
We note that lifetimes are defined as τ ) 1/k. Sono et al. apparently
confused half-lives (t1/2 ) ln 2/k) with lifetimes.
(16) Choi, S. Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard,
S. J .; Newcomb, M.; Putt, D. A.; Upadhyaya, S. P.; Xiong, Y. J . Am.
Chem. Soc. 1996, 118, 6547-6555.
(17) Liu, K. E.; J ohnson, C. C.; Newcomb, M.; Lippard, S. J . J . Am.
Chem. Soc. 1993, 115, 939-947.
(18) Brookhart, M.; Kegley, S. E.; Husk, G. R. Organometallics 1984,
3, 650-652.
(19) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 39, 3815-
3818.
(20) Rodriques, K. E. Tetrahedron Lett. 1991, 32, 1275-1278.

9116 J . Org. Chem., Vol. 62, No. 26, 1997
Toy et al.
Sch em e 2a
diastereomeric purity of 12 prepared from each enantio-
merically enriched acid 8. From integration of these
signals from the amides prepared from the enantiomeri-
cally enriched samples, the sample from (-)-8 had a 99:1
diastereomer ratio, and the sample from (+)-8 had a 97.5:
2.5 diastereomer ratio.
Care was taken to avoid a diastereomeric separation
in preparation of the samples of amide 12, but the
possibility exists that the amide-forming reaction led to
a kinetic resolution. However, such a resolution appar-
ently did not occur on the basis of the observation that
the optical rotations of the samples of resolved acids 8
were consistent with the % ee values assigned from the
NMR spectra of the diastereomeric amides 12. Specifi-
cally, the sample of (+)-8 had [R]25D ) +260.2°, and (-)-8
a
(a) CH2dCHCH2MgBr, THF, 0 °C (86%); (b) PCC, CH2Cl2
(84%); (c) Et3N, THF, reflux (32%).
provide a convenient GC method for assay for 8, the acid
was converted to its methyl ester 9 by treatment with
diazomethane. Probe 3 was prepared from alcohol 6 by
conversion to the methanesulfonate at low temperature
and reduction of the mesylate with lithium triethylboro-
hydride.22
had [R]25 ) -267.9° at comparable concentrations in
D
CHCl3. These two rotations give absolute rotations of
+274° and -273°, respectively, when corrected for the
diastereomeric ratios obtained from the NMR analyses
of amides 12.
The resolved acids 8 were converted to probes 3 by
lithium aluminum hydride reduction to alcohols 6 fol-
lowed by the mesylate formationsreduction protocol used
for preparation of the racemic sample. In these se-
quences, intermediates were not purified, and we assume
that the samples of probe 3 had the same % ee values as
the original samples of acids 8.
The various possible oxidation products from probe 3
were readily separated from one another by GC and had
characteristic mass spectral fragmentation patterns that
permitted conclusive identifications. The availability of
the authentic samples also allowed us to run a number
of control reactions to determine stability of the products
to the enzyme reaction conditions and analytical condi-
tions.
En zym e-Catalyzed Oxidation s. Much of the mecha-
nistic work with cytochrome P450 enzymes has involved
mammalian hepatic enzymes induced by drug treatment.
One of the rat liver enzymes induced by phenobarbital
treatment (CYP2B1)26 is known to be a relatively non-
selective oxidation catalyst and has been employed widely
in mechanistic studies, and that isozyme was employed
in this work.
The initial rearranged oxidation product from probe 3
is alcohol 10 (Scheme 2). Further oxidation of 10 would
give ketone 11a , but this â,γ-unsaturated ketone was
expected to isomerize in the buffer mixture of the enzyme
reactions to give the conjugated isomer 11b. Alcohol 10
was prepared by addition of allylmagnesium bromide to
4-(trifluoromethyl)benzaldehyde.23 Ketone 11 was pre-
pared as a 9:1 mixture of 11a and 11b by pyridinium
chlorochromate (PCC) oxidation of 10. That this was a
mixture of the two alkene isomers was demonstrated by
treatment of the 9:1 mixture with triethylamine in
refluxing tetrahydrofuran to give a 1:19 mixture of 11a
and 11b. These compounds were also found to be
unstable and were prepared immediately before use.
Highly enantiomerically enriched samples of 3 were
prepared from the resolved acids 8 by the method
previously used for preparation of enantiomerically en-
riched samples of 2.11 Thus, acid 8 was converted to its
ethyl ester and partially hydrolyzed with a lipase.
Separation of the acid from the ester and subsequent
saponification of the ester provided the enantiomers of
8 with 84% and 90% ee. The enantiomerically enriched
acids were converted to their dehydroabeitylamine salts
which were recrystallized to effect further enrichment.24
Samples of the recovered acids were converted to amides
12 by reaction with (S)-1-phenylethylamine and 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochlo-
ride,25 and the diastereomeric mixtures were analyzed
by 1H NMR spectroscopy. Samples of the mixtures of
amides in CDCl3 did not display clearly resolved signals
for the diastereomers, but samples in C6D6 did. The
signals for the aryl protons adjacent to the CF3 groups
in the two diastereomers (δ 6.5 and 6.6) had baseline
resolution at 500 MHz and were used to determine the
The hepatic cytochrome P450 enzymes require a re-
ductase enzyme which delivers reducing equivalents from
NADPH to the substrate-enzyme complex and to the
subsequently formed substrate-enzyme-superoxide com-
plex. The reductase enzyme is unstable and undergoes
proteolysis to an inactive form even when stored at -80
°C. Fortunately, the reductase enzyme has been ex-
pressed in E. coli, thereby facilitating the production of
large amounts of enzyme with a significant decrease in
time and cost involved.27 Most of the oxidations con-
(21) Gassman, P. G.; Hodgson, P. K. G.; Balchunis, R. J . J . Am.
Chem. Soc. 1976, 98, 1275-1276.
(22) Brown, H. C.; Kim, S. C.; Krishnamurthy, S. J . Org. Chem.
1980, 45, 1-12.
(23) Yamataka, H.; Nishikawa, K.; Hanafusa, T. Chem. Lett. 1990,
9, 1711-1714.
(26) Nelson, D. R.; Koymans, L.; Kamataki, T.; Stegeman, J . J .;
Feyereisen, R.; Waxman, D. J .; Waterman, M. R.; Gotoh, O.; Coon, M.
J .; Estabrook, R. S.; Gunsalus, I. C.; Nebert, D. W. Pharmacogenetics
1996, 6, 1-42.
(27) Hanna, I. H.; Teiber, J . F.; Kokones, K. L.; Hollenberg, P. F.
Arch. Biochem. Biophys., in press.
(24) Berson, J . A.; Pederson, L. D.; Carpenter, B. K. J . Am. Chem.
Soc. 1976, 98, 122-143.
(25) Sheehan, J . C.; Cruickshank, P. A.; Boshart, G. L. J . Org. Chem.
1961, 26, 2525-2528.

Radical Probe for Enzyme-Catalyzed Hydroxylations
J . Org. Chem., Vol. 62, No. 26, 1997 9117
Ta ble 1. Resu lts of CYP 2B1 Hyd r oxyla tion of P r obe 3
Ta ble 2. Resu lts of P r od u ct Sta bility Con tr ol Rea ction s
substrate concentration
C/Aa
% conversionb turnoverc
% yields of productsb
substrate
(nmol)
(()-3
(+)-3
(-)-3
(()-3
(+)-3
(-)-3
highd
4.6
6.9
4.3
4.0
5.3
2.6
0.9
0.9
0.8
154
145
144
280
280
330
seta
1
6
7
8
10
11
6 (57)
7 (67)
10 (184)
6 (28)
7 (20)
8 (35)
25
20
48
ndc
ndc
lowe
13
14
19
39
2
39
32
29
15
24
64
a
Average ratio of cyclic products (6 + 7) to acyclic product 10.
11 (72)
36
See the Supporting Information for a complete table of results.
Average % conversion of substrate. c Average catalyst turnover.
b
a
The “sets” are a group of experiments performed simulta-
neously with the same batch of cytochrome P450 and reductase
d
Averages from three sets of experiments containing 7.5-14.5
µmol of substrate. e Averages from three sets of experiments con-
taining 1.05-1.30 µmol of substrate.
enzymes. Absolute % yields of products. c nd ) not determined.
b
Ta ble 3. Resu lts of P r od u ct Recover y Stu d ies
ducted in this work employed expressed reductase. In
independent studies, we found that CYP2B1 hydroxyla-
tions of probe 2 conducted with expressed reductase gave
results comparable to those obtained with rat liver
reductase.
experimenta
A
substrate
nmolb
% recoveryc
6
7
10
6
7
10
6
64
28
15
64
28
15
64
28
15
85
68
85
100
85
96
103
77
82
B
C
Racemic probe 3 and both enantiomers of 3 were
oxidized by CYP2B1 in reactions conducted using a
reconstituted system consisting of the cytochrome P450
and reductase enzymes, NADPH, and dilauroyl phos-
phatidylcholine (DLPC). Complete results are given in
the Supporting Information; Table 1 contains a summary
where each listed value is the average from three
independent experiments. The experiments can be di-
vided into two groups depending on the substrate con-
centrations used. In Table 1 we have given the average
ratios of cyclic to acyclic products found in the experi-
ments using high and low substrate concentrations. One
notes that the differences in product ratios for the two
enantiomers of 3 were reproducible.
7
10
a
A mixture of 6, 7, and 10 was studied in each experiment.
The experiments were conducted in a manner similar to the
enzyme oxidations described in the Experimental Section with the
following changes. In A, no enzymes were present. In B, NADPH
was omitted. In C, a fully competent oxidation system containing
12.2 µmol of trans-2-(tert-butoxycarbonyl)-1-methylcyclopropane
b
was employed. Amount of substrate introduced. c Absolute %
yields of recovered products as determined by GC analysis against
a standard.
tions (Table 2). As noted above, aldehyde 7 and ketone
11 were unstable when stored at room temperature. The
observed oxidation products (6, 7, and 10) and putative
products 8 and 11, in the approximate amounts isolated
from the oxidations of 3, were subjected to fully compe-
tent enzyme oxidation conditions. In the reaction of
alcohol 6, aldehyde 7 and acid 8 were obtained, and acid
8 also was obtained from oxidation of aldehyde 7.
The only products observed in the enzyme catalyzed
oxidations of 3 were the cyclic alcohol 6, the aldehyde 7,
and the ring-opened alcohol 10. Acid 8 and ketone 11
were not detected at a limit of about 1% relative to the
amounts of products observed. The enzyme turnover
values were a function of the concentrations of substrate
3 present, but turnover numbers in oxidations of the two
enantiomers of 3 were similar when the enantiomers
were present in comparable amounts. Importantly, we
did not observe products at GC retention times expected
for phenols from 3.
Another set of control reactions was conducted to
determine the extent of product recovery expected from
the reactions of 3 (Table 3). Thus, mixtures of products
6, 7, and 10 were treated with the fully competent
enzyme system and a test substrate, with the enzyme
system lacking NADPH, and with the reaction mixture
lacking the enzymes. Product recoveries of 6 and 10 were
high in each of these reactions. Unstable aldehyde 7 was
recovered in good to high yield.
The formation of aldehyde 7 in the cytochrome P450
oxidation of probe 3 is noteworthy. Oxidation of alcohol
6 to aldehyde 7 in the control reactions demonstrated
that 6 was a substrate, but we and others had not
previously observed aldehyde formation when methyl-
cyclopropane probes were oxidized by CYP2B1 in low
The total amount of oxidized substrate was 0.6-2.5%
in the high substrate concentration experiments and 11-
23% in the low concentration experiments. Interestingly,
the enzyme turnover values were larger when substrate
3 was present in low concentrations. This effect could
arise from an inhibitory property of the substrate for the
enzyme, but it might have a more mundane origin. The
isozyme is membrane-bound in nature, and the probe at
higher concentrations might have disrupted the micelles
in the reconstituted system. In any event, the amount
of enantiomerically enriched substrate oxidized in the low
substrate concentration experiments far exceeded the
amount of minor enantiomer contamination in the en-
riched samples thus ensuring that we were observing
products from hydroxylation of each enantiomer.
conversion experiments.8,10-12,14
Nevertheless, Zaks and
Dodds reported that probe 2 was oxidized to the cyclic
aldehyde and cyclic acid in addition to the cyclic alcohol
by chloroperoxidase from the fungus Caldaromyces fum-
ago, a heme-containing enzyme that is closely related to
cytochrome P450 enzymes.28 Apparently, in comparison
to hydrocarbon probe 3, alcohol 6 is selectively bound and/
or oxidized by CYP2B1. It is possible that the second
A series of control reactions was performed to deter-
mine the stability of the products to the reaction condi-
(28) Zaks, A.; Dodds, D. R. J . Am. Chem. Soc. 1995, 117, 10419-
10424.

9118 J . Org. Chem., Vol. 62, No. 26, 1997
Toy et al.
oxidation of alcohol 6 occurred in competition with release
of this product from the enzyme.
tion mechanism of cytochrome P450 catalyzed hydroxy-
lation even though it suffers from the same limitation
as most of the other probes that have been employed in
such studies in that both radical and cationic cyclopro-
pylcarbinyl species will fragment in the same manner.
In independent studies that will be reported in detail
later, we have measured rate constants for ring opening
of the radical 18 using benzeneselenol trapping as a
competing reaction,30 the same method as employed in
calibrations of ring opening rate constants for other
2-arylcyclopropylcarbinyl radicals.29,31 The rate constant
for ring opening of 18 at 37 °C is 9 × 1011 s-1, about three
times greater than that for ring opening of parent radical
16. The average ratio of cyclic to ring-opened products
obtained with probe 3 was about 4.5, which requires that
the minimum limit for the rate constant for radical
capture in the hydroxylation is 4 × 1012 s-1 at 37 °C.
Thus, the lifetime of the radical must be 250 fs or less at
this temperature. The minimum rate constant for cap-
ture is only slightly smaller than the rate constant for
decomposition of a transition state calculated from
conventional transition state theory (6 × 1012 s-1), and
the free energy of activation for radical capture, a
defining requirement for an intermediate, must be less
than 0.3 kcal/mol. We emphasize that the 250 fs lifetime
is the limiting case wherein one assumes that no cationic
rearrangement occurred.
The discussion in the above paragraph is founded on
the premise that the radical rearrangement rate con-
stants are not strongly influenced by the enzyme. The
studies of hydroxylations of the two enantiomers of probe
3 provide additional evidence that this assumption is
reasonable. As was the case with hydroxylation of the
enantiomers of probe 2,11 the product ratios for the
enantiomers of 3 were only slightly (but reproducibly, see
below) different. On the basis of the observation that
the constrained radicals 13-15 fragment with nearly the
same rate constants, one must conclude that truly
dramatic enzymic constraint would be required to reduce
the rate constants for ring opening of the radicals by more
than 2 orders of magnitude necessary to bring the results
into alignment with a radical lifetime in the enzyme-
catalyzed hydroxylation of 70000 fs (the value one
calculates from the results of hydroxylation of bicyclo-
[2.1.0]pentane8). The likelihood that the chiral enzyme
could have such a dramatic effect on the rate constants
for fragmentation of both of the enantiomeric radicals 18
seems remote as an independent fact. Taken together
with the other evidence that the enzyme does not
influence the cyclopropylcarbinyl radical ring-opening
rate constants strongly, we believe the premise that such
an effect occurs is unreasonable.
Discu ssion
The oxygen rebound mechanism for cytochrome P450
catalyzed hydroxylation of an unactivated C-H position
is shown in path a in Figure 1. In this mechanism, a
radical is formed as a discrete, albeit possibly short-lived,
intermediate. From the results with probe 1, we con-
cluded that a discrete radical intermediate could not be
formed.14 The lifetime15 of the “radical” was only about
70 fs, which is too short a lifetime for an intermediate,
and the “radical” could only be a component of the
transition state in a concerted insertion process (Figure
1, path b). On the basis of the observation that cation-
derived products were formed in the hydroxylation of
probe 1, we reasoned that previous studies with radical
probes that gave rearranged products gave misleading
results concerning the radical lifetime. Specifically, we
suggested that in cases where the skeletal reorganization
occurring for radical and cationic rearrangements is the
same, that the rearranged products result from both
species. Two possible routes to cationic species are
discussed later.
Our mechanistic conclusions are open to criticism. One
important point that must be considered is the possibility
that the enzyme can dramatically alter the rate constants
for ring opening of the radical. In fact, we specifically
raised this point in an early report of the application of
hypersensitive radical probe 2 in hydroxylations cata-
lyzed by a methane monooxygenase system.17 If the
enzyme affects the radical ring opening rate constants,
presumably by steric compression that forces the aro-
matic ring into poor alignment with the breaking C-C
bond of the cyclopropyl ring, then the quantitative
aspects of the studies are vitiated. However, a prepon-
derance of evidence suggests that any influence of the
enzyme on the rate constants for ring opening of cyclo-
propylcarbinyl radicals can only be minor. Among the
more important observations in this regard are the facts
(1) that enforced nonoptimal alignments of the aromatic
ring with the breaking C-C bond in radicals 13-15
result in only minor differences in the rate constants for
the fragmentation reactions in comparison to the parent
radical 16,29 (2) that the constrained probe 17, in which
the aryl ring cannot be twisted appreciably by the
enzyme, is hydroxylated by cytochrome P450 with an
apparent radical lifetime of 80 fs,12 and (3) that both
enantiomers of probe 2, which should interact differently
with the chiral enzyme, give similar product ratios in
cytochrome P450 hydroxylation studies.11
If the mechanistic model for cytochrome P450 hydrox-
ylation we have proposed, an insertion process with a
competing pathway to cationic species, is reasonable,
then the question of the origin of the cationic species
remains unresolved. The following possible routes to
cation formation have been considered (Figure 2): (1) an
oxidation event competes with collapse of the “radical”
in the transition state of the hydroxylation and (2) the
first formed product is not an alcohol but rather a
protonated alcohol formed by insertion of “OH+” into the
C-H bond instead of “O”, and proton transfer from the
The mechanistic probe we are testing in this work,
compound 3, provides supporting evidence for the inser-
(30) Choi, S.-Y.; Toy, P.; Newcomb, M. Unpublished results.
(31) Newcomb, M.; J ohnson, C. C.; Manek, M. B.; Varick, T. R. J .
Am. Chem. Soc. 1992, 114, 10915-10921.
(29) Martin-Esker, A. A.; J ohnson, C. C.; Horner, J . H.; Newcomb,
M. J . Am. Chem. Soc. 1994, 116, 9174-9181.

Radical Probe for Enzyme-Catalyzed Hydroxylations
J . Org. Chem., Vol. 62, No. 26, 1997 9119
nated alcohol and hydroxide ligated to iron. Rapid proton
transfer to hydroxide would give the alcohol product and
water. The attraction of this pathway is that one could
postulate that aryl-substituted cyclopropane probes would
have an anchimeric assistance route for ionic fragmenta-
tion not available to the simple cyclopropane probes (i.e.
19). In addition, there is computational support for a
low energy pathway for insertion of OH+ into C-H bonds
from simple models for the heme-iron-H2O2 complex
such as the hydroperoxonium ion (H2O-OH)+;34 one
awaits advanced computations of reactions of an Fe-
H2O2 complex. The problem with this pathway is that it
apparently requires that the insertion occurs before loss
of water from the enzyme-H2O2 complex as shown in
Figure 2. Although the precise timing of the oxidation
step relative to loss of water in a cytochrome P450
hydroxylation has not been established, it is commonly
thought that the actual oxidizing entity is an iron-oxo
species similar to compound I observed in decompositions
of H2O2 by peroxidase enzymes; that is, loss of water
precedes oxidation.
F igu r e 2. Two possible routes for cation formation in cyto-
chrome P450 hydroxylation. In the upper scheme, electron-
transfer competes with collapse in the transition state, and
the alkyl cation formed by oxidation can rearrange. In the
lower scheme, hydroxylation occurs before release of water,
and the first-formed oxidation product is a protonated alcohol
that can rearrange in competition with proton transfer.
protonated alcohol competes with loss of water in a
solvolytic-type reaction. Taken with previous observa-
tions, the results of the present study appear to be more
consistent with the latter pathway as discussed below,
and this ultimately leads to a conclusion that the active
oxidant in the cytochrome P450 hydroxylation might be
an iron-hydrogen peroxide complex (as shown in Figure
2) rather than the commonly assumed iron-oxo inter-
mediate. We caution that such a conclusion is specula-
tive and that our results are only suggestive in this
regard, but we believe the point warrants presentation
on the basis of the current interest in the mechanism of
cytochrome P450 hydroxylation.
There is some evidence that cytochrome P450 enzymes
with iron bound to an alkoxy or hydroxyl group can
oxidize a carbon radical to a cation. For example,
prostacyclin (PGI2) and thromboxane (TxA2) synthases
are cytochrome P450 enzymes that might produce their
respective products from prostaglandin PGH2 by routes
that include oxidations of carbon radical intermediates
by iron-alkoxy moieties.32 In a similar manner, an iron-
hydroxy species might oxidize a DNA radical to a cation
in one of the competing reactions resulting in DNA strand
scission by the anticancer agent bleomycin.33 To accom-
modate such an oxidation with the results of many
cyclopropane probe studies, however, one would need to
rationalize that oxidation of a cyclopropylcarbinyl radical
with an aryl group at C2 is considerably more favorable
than oxidation of a simple cyclopropylcarbinyl radical,
which seems to be unlikely. This rationalization is
necessary because simple cyclopropane probes such as
methylcyclopropane are hydroxylated at the methyl
position without formation of ring-opened products.8,9 An
important point casting further doubt on the radical
oxidation route to a cation is the report that cytochrome
P450 hydroxylation of isopropylcyclopropane to the ter-
tiary alcohol occurs without formation of rearranged
products;9 the putative tertiary cation from this species
is much more stable than the primary cation that would
have to be produced from methylcyclopropane probes.
In the alternative route to rearranged products, inser-
tion of “OH+” into the C-H bond, the first formed
products of the hydroxylation process would be a proto-
If one again assumes that the insertion model of Figure
1, path b is correct and further assumes that the radical
lifetime in the transition state for P450 insertion is
essentially constant for hydroxylation of any probe, then
the amount of rearranged products obtained in probe
studies can be divided into the amount resulting from
the radical process and that from the ionic process. On
the basis of the rate constants for ring opening of the
cyclopropylcarbinyl radicals, three times as much rear-
ranged product should have been found from hydroxyl-
ation of probe 3 as was found in the oxidations of probe
2, but, in fact, more acyclic product was obtained in the
oxidations of probe 2. This qualitative observation
indicates that the ionic route to rearranged products was
more important in the case of probe 2.
One can attempt to quantitate the results by assuming
that the radical capture rate constant in the hydroxyla-
tion insertion transition state is 1.5 × 1013 s-1, the value
obtained in study of probe 1. In that case, the ratio of
cyclic to acyclic products resulting solely from the radical
route would be 97:3 for probe 2 and 92:8 for probe 3.
Hydroxylation of probe 2 gave ratios of cyclic to acyclic
products that averaged about 75:25,11 and the ratio found
in this work for probe 3 averaged about 82:18. Thus, the
amount of ionic rearrangement that apparently occurred
with probe 2 was about 2.5 times as great as that with
probe 3.
Both of the possible pathways we envision for produc-
tion of cationic products (Figure 2) could exhibit a
response to the trifluoromethyl substituent on the aro-
matic ring. This substituent has a stabilizing effect on
a radical (witness the 3-fold acceleration in ring opening
of radical 18 relative to radical 16), and it is strongly
destabilizing for a cation. Nevertheless, a remote effect
on the oxidation potential of the radical center on the
methylene group in 18 (as opposed to the benzylic radical
center in the rearranged product) might be expected to
(32) Ullrich, V.; Brugger, R. Angew. Chem., Int. Ed. Engl. 1994, 33,
1911-1919.
(33) Stubbe, J .; Kozarich, J . W. Chem. Rev. (Washington, D.C.) 1987,
87, 1107-1136.
(34) Bach, R. D.; Mintcheva, I.; Estevez, C. M.; Schlegel, H. B. J .
Am. Chem. Soc. 1995, 117, 10121-10122.

9120 J . Org. Chem., Vol. 62, No. 26, 1997
Toy et al.
be quite small, and we have already noted that the
radical oxidation pathway is inconsistent with the ob-
servation that hydroxylation of the secondary and ter-
tiary positions of simple alkylcyclopropanes proceeds with
no rearrangement.9,10 Alternatively, the rate of the aryl-
assisted solvolysis of protonated alcohol (i.e. 19) should
be reduced by the CF3 group. On balance, the results
appear to be more in accord with a solvolysis pathway
than a radical oxidation pathway. This leads us to the
speculation that water dissociation from the enzyme-
H2O2 complex might not precede the oxidation event.
Finally, we comment on the slight differences in
product ratios observed for the two enantiomers of 3.
Similar small but reproducible differences in product
ratios were also found in hydroxylation of probe 2.11 We
argued above that drastic effects of the enzyme on the
unimolecular radical rearrangement rate constants are
not expected; essentially, unimolecular collapse of the
transition state ensemble competes with unimolecular
radical rearrangement, and neither process should be
influenced significantly by the enzyme. However, there
is little reason to believe that minor kinetic effects for
cation production will not be found in the diastereomeric
ensembles of the two enantiomers with the chiral enzyme
because one of the competing reactions in each possible
route to the cation is “bimolecular” in nature. If radical
oxidation occurs, then the electron transfer must compete
with the collapse of the transition structure that results
in alcohol product. If assisted solvolysis of a protonated
alcohol is the origin of cationic products, then this
solvolysis step must compete with proton transfer from
protonated alcohol to hydroxide bound to iron. The
kinetics of both electron transfer and proton transfer
events will be affected by the distances between the
reactive centers. Thus, we propose that differences in
the assembly of the diastereomeric enzyme-substrate
complexes result in small differences in the distances
between reactive centers that ultimately affect the rate
of a competition reaction and thus the amount of cation-
derived products.
leads to speculation that the enzyme-H2O2 complex
might be the active oxidizing moiety rather than the
“iron-oxo” intermediate formed by loss of water from the
complex.
Exp er im en ta l Section
Gen er a l Meth od s. Commercially available reagents were
purchased form either the Sigma or Aldrich Chemical Co.
unless otherwise noted 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 were distilled under a nitrogen
atmosphere over sodium and benzophenone ketyl. Methylene
chloride was distilled under a nitrogen atmosphere over
phosphorus pentoxide. Dimethyl sulfoxide (DMSO) was dis-
tilled in vacuo from calcium hydride.
NMR spectra were acquired on either a Varian Gemini 300
or a General Electric QE-300 spectrometer. Gas chromatog-
raphy analyses were performed using flame ionization detec-
tion on a Varian 3400 chromatograph (15 m × 0.54 mm bonded
phase Carbowax column, Alltech). Gas chromatography/mass
spectral (GC/MS) analyses were performed using a Hewlett-
Packard Model 5890 GC interfaced to a Hewlett-Packard
Model 5971 mass selective detector (30 m × 0.25 mm capillary
bonded phase Carbowax column, Alltech). High-resolution
mass spectral analyses were performed by the Central Instru-
mentation Facility at Wayne State University (Detroit, MI).
Melting points were determined using a Unimelt capillary
melting point apparatus (Thomas-Hoover) and are uncor-
rected. Radial chromatography was performed on a Chroma-
totron Model 7294T (Harrison Research Corp.) using plates
coated with TLC grade silica gel (Merck) with gypsum binder
and fluorescent indicator.
En zym a tic oxid a tion r ea ction s were performed in re-
constituted systems using the purified isozyme CYP2B135 and
expressed reductase27 at 37 °C in 50 mM potassium phosphate
buffer, pH 7.4, containing 1 mM desferrioxamine and 0.075
M KCl, with a total reaction volume of 2 mL. A mixture of
0.6 nmol of CYP2B1 and 1.2 nmol of reductase in buffer was
allowed to stand on ice for 5 min. A suspension of DLPC (0.96
nmol) in buffer was sonicated. The enzyme and micelle
mixtures were combined, and this mixture was allowed to
stand on ice for 5 min. The mixture was diluted with buffer,
and the substrate was added either neat (high substrate
concentration) or as a solution in ca. 10 µL of methanol (low
substrate concentration). The reaction mixture was incubated
for 2 min at 37 °C, and the reaction was initiated by the
addition of NADPH (final concentration of 1.2 mM). The
reaction mixture was shaken gently at 37 °C for 30 min and
then extracted with methylene chloride (3 × 2 mL). The
combined organic phases were 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 using gas chromatography with flame ionization
detection, and product identities were determined by compari-
son with authentic samples using selected ion monitoring
(SIM) GC/MS analysis.
Con clu sion s
The hypersensitive radical probe 3 containing the
p-(trifluoromethyl)phenyl group functions well in studies
of hydroxylations by the cytochrome P450 isozyme
CYP2B1. Good yields of products from oxidation at the
methyl position were obtained from both enantiomers of
3, and aromatic ring oxidation apparently did not occur
to an appreciable extent. The similar product ratios
obtained from the two enantiomers of 3 provides more
evidence that the enzyme does not have a significant
effect on the rate constants for radical rearrangements
of aryl-substituted cyclopropylcarbinyl radicals. The
small amount of rearranged product formed is consistent
with an insertion pathway for the enzyme-catalyzed
hydroxylation with a competing process that involves
production of a cationic species. From comparison of the
amount of apparent cation-derived product produced in
hydroxylation of 3 to that produced in hydroxylation of
the analogous probe not containing the trifluoromethyl
substituent (2), a solvolytic-type pathway with anchi-
meric assistance from the aromatic group is suggested
for production of the cation-derived rearranged products.
Such a pathway suggests that the first formed species
in the hydroxylation reaction is a protonated alcohol
formed by “OH+” insertion into a C-H bond, and this
Control reactions in Table 2 were performed by dissolving
the substrates in methanol and adding aliquots of these
solutions to two reaction mixtures prepared as above, one
reaction mixture containing enzymes and one without the
enzymes. The reactions were conducted and extracted as
above. The amounts of recovered material were determined
by comparison of the ratios of the GC areas of the compounds
of interest to those of standards in the two reactions.
N-Meth oxy-N-m eth yl-tr a n s-4-(tr iflu or om eth yl)cin n am -
a m id e (4). A solution of 4-(trifluoromethyl)benzaldehyde (10.0
g, 57.4 mmol) and N-methoxy-N-methyl-2-(triphenylphospho-
ranylidene)acetamide (23.1 g. 63.6 mmol) in CH2Cl2 (150 mL)
was stirred at room temperature for 16 h. The solvent was
(35) Saito, T.; Strobel, H. W. J . Biol. Chem. 1981, 256, 984-988.

Radical Probe for Enzyme-Catalyzed Hydroxylations
J . Org. Chem., Vol. 62, No. 26, 1997 9121
then removed in vacuo, and the crude product was chromato-
graphed on silica gel (40% ethyl acetate in hexanes) to afford
4 (14.1 g, 54.3 mmol, 95%) as a white solid. Mp 50-52 °C. 1H
NMR (CDCl3): δ 3.32 (3H, s), 3.78 (3H, s), 7.10 (1H, d, J )
15.9 Hz), 7.65 (4H, dd, J 1 ) 12.0 Hz, J 2 ) 9.0 Hz), 7.74 (1H, d,
J ) 15.9 Hz). 13C NMR (CDCl3): δ 32.5, 61.9, 123.8 (q, J C-F
) 276.9 Hz), 125.6 (2C, q, J C-F ) 3.7 Hz), 128.1 (2C), 131.3 (q,
J C-F ) 33.1 Hz), 138.6, 141.5, 166.2. HRMS: calcd for
C12H12F3NO2, 259.0812; found, 259.0820.
(2H, d, J ) 8.1 Hz), 9.36 (1H, d, J ) 4.2 Hz). 13C NMR
(CDCl3): δ 16.7, 25.9, 33.7, 124.1 (q, J C-F ) 270.2 Hz), 125.5
(2C, q, J C-F ) 4.4 Hz), 126.5 (2C), 128.9 (q, J C-F ) 32.0 Hz),
143.3, 199.1. MS: calcd for C11H9F3O, 214; (M)+ found at m/e
) 214.
tr a n s-2-(4-(Tr iflu or om eth yl)p h en yl)cyclop r op a n eca r -
boxylic Acid (8). A suspension of 5 (7.71 g, 28.2 mmol) and
potassium tert-butoxide (17.11 g, 152 mmol) in ether (200 mL)
and water (1.0 mL, 55.6 mmol) was stirred at room temper-
ature for 16 h. The mixture was acidified by the slow addition
of concentrated HCl, and the aqueous mixture was extracted
with CH2Cl2 (3 × 200 mL). The combined organic layers were
washed with brine (200 mL), dried over Na2SO4, filtered, and
concentrated in vacuo to afford 8 (5.85 g, 25.4 mmol, 90%) as
a white solid. Mp 110-111 °C. 1H NMR (CDCl3): δ 1.45 (1H,
N -Me t h oxy-N -m e t h yl-t r a n s-2-(4-(t r iflu or om e t h yl)-
p h en yl)cyclop r op a n eca r boxa m id e (5). A solution of tri-
methylsulfoxonium iodide (24.1 g, 110 mmol) in DMSO (100
mL) was cooled with a room-temperature water bath, and NaH
(2.6 g, 108 mmol) was added portion wise over 15 min. After
the addition was complete, the suspension was allowed to stir
for 1 h, during which time it became homogeneous. A solution
of 4 (14.1 g, 54.3 mmol) in DMSO (50 mL) was added via
cannula, and the reaction mixture was stirred for 6 h. The
reaction mixture was quenched by pouring it into saturated
aqueous NH4Cl soln (500 mL). The mixture was extracted
with CH2Cl2 (3 × 250 mL). The combined organic layers were
washed with brine (200 mL), dried over MgSO4, filtered, and
concentrated in vacuo. The crude material was chromato-
graphed on silica gel (40% ethyl acetate in hexanes) to afford
5 (12.8 g, 46.8 mmol, 86%) as a clear, colorless oil. 1H NMR
(CDCl3): δ 1.30 (1H, ddd, J 1 ) 8.4 Hz, J 2 ) 6.3 Hz, J 3 ) 4.5
Hz), 1.65 (1H, ddd, J 1 ) 9.0 Hz, J 2 ) 5.4 Hz, J 3 ) 4.2 Hz),
2.38-2.46 (1H, m), 2.50 (1H, ddd, J 1 ) 9.3 Hz, J 2 ) 6.6 Hz, J 3
) 4.2 Hz), 3.19 (3H, s), 3.65 (3H, s), 7.18 (2H, d, J ) 9.0 Hz),
7.48 (2H, d, J ) 9.0 Hz). 13C NMR (CDCl3): δ 16.5, 21.8, 25.2,
32.4, 61.5, 124.1 (q, J C-F ) 270.3 Hz), 125.2 (2C, q, J C-F ) 3.3
Hz), 126.3 (2C), 128.3 (q, J C-F ) 32.4 Hz), 145.0, 172.2.
HRMS: calcd for C13H14F3NO2, 273.0977; found, 273.0974.
(tr a n s-2-(4-(Tr iflu or om eth yl)ph en yl)cyclopr opyl)m eth -
a n ol (6). A solution of 5 (2.0 g, 7.3 mmol) in THF (75 mL)
was cooled to 0 °C, and LiAlH4 (0.5 g, 20.8 mmol) was added.
The mixture was stirred for 2 h, after which time the reaction
mixture was quenched by the sequential addition of water (0.5
mL), 15% NaOH (0.5 mL), and water (1.5 mL). After stirring
for an additional 2 h, the suspension was filtered, and the
filtrate was washed with additional THF (100 mL). The
combined organic solutions were concentrated in vacuo, and
the resulting residue was dissolved in dry THF (75 mL). This
mixture was cooled to 0 °C, and LiAlH4 (0.5 g, 20.8 mmol) was
added. This mixture was stirred for 2 h, the reaction was
quenched, and the crude product was isolated as above. The
crude product was chromatographed on silica gel (40% ethyl
acetate in hexanes) to afford 6 (1.0 g, 4.5 mmol, 62%) as a white
solid. Mp 61 °C. 1H NMR (CDCl3): δ 1.01-1.06 (2H, m), 1.41
(1H, bs), 1.46-1.57 (1H, m), 1.87-1.93 (1H, m), 3.66 (2H, d, J
) 6.6 Hz), 7.17 (2H, d, J ) 8.4 Hz), 7.52 (2H, d, J ) 8.4 Hz).
ddd, J 1 ) 8.4 Hz, J 2 ) 6.6 Hz, J 3 ) 4.8 Hz), 1.73 (1H, dt, J 1
)
9.3 Hz, J 2 ) 5.1 Hz), 1.96 (1H, ddd, J 1 ) 9.0 Hz, J 2 ) 5.4 Hz,
J 3 ) 4.2 Hz), 2.65 (1H, ddd, J 1 ) 9.3 Hz, J 2 ) 6.6 Hz, J 3 ) 3.9
Hz), 7.21 (2H, d, J ) 8.4 Hz), 7.55 (2H, d, J ) 8.4 Hz), 12.08
(1H, bs). 13C NMR (CDCl3): δ 17.6, 24.2, 26.5, 124.1 (q, J C-F
) 270.2 Hz), 125.5 (2C, q, J C-F ) 3.3 Hz), 126.5 (2C), 129.0 (q,
J C-F ) 33.1 Hz), 143.6, 179.4. HRMS: calcd for C11H9F3O2,
230.0555; found, 230.0560.
The enantiomerically enriched carboxylic acids 8 were
prepared by P-30 Lipase36 (Amano International Enzyme Co.,
from Pseudomonas cepacia, formerly classified as Pseudomo-
nas fluorescens) hydrolysis of the corresponding ethyl esters
by the method previously reported.11 The reaction was ap-
proximately 50% complete after 8 days. From 9.0 g (34.9
mmol) of the ester, 2.0 g (8.7 mmol) of acid and 4.2 g (16.3
mmol) of ester were isolated following the standard workup.
The ester was saponified, and the acid was isolated. Three
recrystalizations of the acids as their dehydroabietylamine
salts (ethyl acetate) afforded optically enriched acids 8. The
enantiomeric excesses were determined by derivatizing the
acids with (S)-1-phenylethylamine, 1-(3-(dimethylamino)pro-
pyl)-3-ethylcarbodiimide hydrochloride, and 4-(dimethylami-
no)pyridine in CH2Cl2. 1H NMR analysis (500 MHz, C6D6) of
the resulting amides showed that the diastereomeric mixtures
were 97.5:2.5 and 1:99. The optical rotations of the acids were
[R]25 ) +260.2° (c ) 2.26, CHCl3) and [R]25 ) -267.9° (c )
D
D
2.18, CHCl3).
tr a n s-1-Meth yl-2-(4-(tr iflu or om eth yl)p h en yl)cyclop r o-
p a n e (3). A solution of 6 (0.284 g, 1.31 mmol) in THF (8 mL)
was cooled to -30 °C. To this was added sequentially via
syringe triethylamine (0.45 mL, 3.23 mmol) and methane-
sulfonyl chloride (0.11 mL, 1.42 mmol). The mixture was
stirred at -30 °C for 30 min and then cooled to -78 °C.
A
solution of lithium triethylborohydride (1.0 M in THF, 4.0 mL,
4.0 mmol) was added via syringe. The mixture was allowed
to warm slowly to room temperature and was stirred for 13 h.
The reaction mixture was quenched by the addition of 30%
H2O2 (2 mL) and 15% aqueous NaOH (2 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 material was purified by radial chromatography (pen-
tane) to afford 3 (0.209 g, 1.04 mmol, 79%) as a clear, colorless
13C NMR (CDCl3): δ 14.3, 21.1, 25.9, 66.0, 124.3 (q, J C-F
)
269.2 Hz), 125.2 (2C, q, J C-F ) 3.5 Hz), 125.9 (2C), 127.8 (q,
J C-F ) 33.1 Hz), 146.9. HRMS: calcd for C11H11F3O2, 216.0762;
found, 216.0759. The enantiomerically enriched alcohols 6
from (+)-8 and (-)-8, prepared from the acids by LiAlH4
reduction as above, were obtained in 77% and 83% yields,
respectively.
(tr a n s-2-(4-(Tr iflu or om eth yl)ph en yl)cyclopr opyl)m eth -
a n a l (7). A solution of 5 (1.13 g, 4.1 mmol) in CH2Cl2 (25 mL)
was cooled to -78 °C, and a solution of DIBALH (1.5 M in
toluene, 4.0 mL, 6.0 mmol) was added via syringe over 5 min.
The mixture was stirred for 3 h, and then methanol (5 mL)
was added. The reaction mixture was allowed to warm slowly
to room temperature and was then diluted with ether (100
mL). The ethereal solution was then washed successively with
10% aqueous HCl soln (100 mL), saturated aqueous NaHCO3
soln (100 mL), and brine (100 mL). The organic layer was then
dried over MgSO4, filtered, and concentrated in vacuo. The
crude product was chromatographed on silica gel (20% ethyl
acetate in hexanes) to afford 7 (0.70 g, 3.3 mmol, 79%) as a
clear, colorless oil that decomposed on standing. 1H NMR
(CDCl3): δ 1.54 (1H, ddd, J 1 ) 8.7 Hz, J 2 ) 6.9 Hz, J 3 ) 5.4
Hz), 1.77 (1H, dt, J 1 ) 9.3 Hz, J 2 ) 5.4 Hz), 2.21 (1H, ddd, J 1
) 8.7 Hz, J 2 ) 5.4 Hz, J 3 ) 4.5 Hz), 2.66 (1H, ddd, J 1 ) 9.3
Hz, J 2 ) 6.6 Hz, J 3 ) 3.9 Hz), 7.21 (2H, d, J ) 8.1 Hz), 7.53
oil. 1H NMR (CDCl3): δ 0.80-0.86 (1H, m), 0.94 (1H, dt, J 1
)
8.1 Hz, J 2 ) 4.8 Hz), 1.06-1.16 (1H, m), 1.20 (3H, d, J ) 5.7
Hz), 1.62 (1H, dt, J 1 ) 8.7 Hz, J 2 ) 4.5 Hz), 7.10 (2H, d, J )
8.4 Hz), 7.48 (2H, d, J ) 8.4 Hz). 13C NMR (CDCl3): δ 18.3,
18.9, 18.9, 24.2, 124.6 (q, J C-F ) 270.2 Hz), 125.1 (2C, q, J C-F
) 2.9 Hz), 125.6 (2C), 127.4 (q, J C-F ) 31.6 Hz), 148.6.
HRMS: calcd for C11H11F3, 200.0813; found, 200.0808. The
enantiomerically enriched samples of compounds (+)-3 and
(-)-3 were synthesized from the corresponding methanols (6)
by the same method and obtained in 80% and 86% yields,
respectively.
Meth yl tr a n s-2-(4-(Tr iflu or om eth yl)p h en yl)cyclop r o-
p a n eca r boxyla te (9). To a solution of 5 (0.25 g, 1.1 mmol)
(36) Xie, Z. F. Tetrahedron: Asymmetry 1991, 2, 733-750.

9122 J . Org. Chem., Vol. 62, No. 26, 1997
Toy et al.
in CH2Cl2 (50 mL) was added dropwise a solution of diazo-
methane in ether until a slight yellow color persisted. The
mixture was stirred open to the atmosphere for 6 h. The
solvent was removed in vacuo, and the crude material was
chromatographed on silica gel (10% ethyl acetate in hexanes)
The ratio of the product mixture was determined by gas
chromatography. This mixture was then dissolved in THF (25
mL) and triethylamine (0.5 mL), and the solution was heated
at reflux for 14 h. The solution was concentrated in vacuo,
and the crude material was purified by radial chromatography
(15% ethyl acetate in hexanes) to afford a 1:19 mixture of 11a
and 11b (0.14 g, 0.654 mmol, 32%). Compounds 11 decom-
posed on standing.
1
to afford 7 (0.25 g, 1.0 mmol, 94%) as a clear, colorless oil. H
NMR (CDCl3): δ 1.34 (1H, ddd, J 1 ) 8.4 Hz, J 2 ) 6.3 Hz, J 3
)
4.5 Hz), 1.65 (1H, ddd, J 1 ) 9.0 Hz, J 2 ) 5.4 Hz, J 3 ) 4.8 Hz),
1.94 (1H, ddd, J 1 ) 8.4 Hz, J 2 ) 5.4 Hz, J 3 ) 4.2 Hz), 2.56
(1H, ddd, J 1 ) 9.3 Hz, J 2 ) 6.3 Hz, J 3 ) 3.9 Hz), 3.72 (3H, s),
7.17 (2H, d, J ) 8.4 Hz), 7.51 (2H, d, J ) 8.4 Hz). 13C NMR
(CDCl3): δ 17.1, 24.2, 25.7, 51.9, 124.1 (q, J C-F ) 270.2 Hz),
125.4 (2C, q, J C-F ) 3.3 Hz), 126.4 (2C), 128.7 (q, J C-F ) 32.0
Hz), 144.2, 173.2. HRMS: calcd for C12H11F3O2, 244.0711;
found, 244.0715.
Characterization data for 11a : 1H NMR (CDCl3): δ 3.78
(2H, dt, J 1 ) 6.6 Hz, J 2 ) 1.2 Hz), 5.20-5.29 (2H, m), 6.01-
6.14 (1H, m), 7.74 (2H, d, J ) 8.4 Hz), 8.07 (2H, d, J ) 8.4
Hz). MS: calcd for C11H9F3O, 214; (M)+ found at m/e ) 214.
Characterization data for 11b: 1H NMR (CDCl3): δ 2.02
(3H, dd, J 1 ) 7.2 Hz, J 2 ) 1.8 Hz), 6.87 (1H, dq, J 1 ) 15.6 Hz,
J 2 ) 1.8 Hz), 7.12 (1H. dq, J 1 ) 15.6 Hz, J 2 ) 6.6 Hz), 7.72
(2H, dd, J 1 ) 8.7 Hz, J 2 ) 0.6 Hz), 8.00 (2H, dd, J 1 ) 8.7 Hz,
J 2 ) 0.6 Hz). MS: calcd for C11H9F3O, 214; (M)+ found at m/e
) 214.
1-(4-(Tr iflu or om eth yl)p h en yl)bu t-3-en e-1-ol (10) was
prepared by the previously reported method.23 1H NMR
(CDCl3): δ 2.14 (1H, d, J ) 3.3 Hz), 2.41-2.60 (2H, m), 4.79-
4.84 (1H, m), 5.15-5.22 (2H, m), 5.73-5.87 (1H, m), 7.48 (2H,
d, J ) 8.4 Hz), 7.61 (2H, d, J ) 8.4 Hz). 13C NMR (CDCl3): δ
43.5, 72.66, 118.48, 124.1 (q, J C-F ) 270.4 Hz), 125.1 (2C, q,
J C-F ) 3.2 Hz), 126.0 (2C), 129.5 (q, J C-F ) 32.4 Hz), 133.6,
147.8.
1-(4-(Tr iflu or om eth yl)ph en yl)bu t-3-en e-1-on e (11a) an d
tr a n s-1-(4-(tr iflu or om eth yl)p h en yl)bu t-2-en -1-on e (11b).
A solution of 10 (0.53 g, 2.45 mmol) and PCC (1.00 g, 4.65
mmol) in CH2Cl2 (25 mL) was stirred at room temperature for
8 h. The suspension was then diluted with ether (75 mL) and
filtered through a pad of silica gel. The organic solution was
concentrated in vacuo, and the crude material was purified
by radial chromatography (15% ethyl acetate in hexanes) to
afford a 9:1 mixture of 11a and 11b (0.44 g, 2.05 mmol, 84%).
Su p p or tin g In for m a tion Ava ila ble: Table of results from
CYP2B1 hydroxylation of probe 3, 1H and 13C NMR spectra of
1
3-10, and H NMR spectra of mixtures of 11a /11b (19 pages).
This material is contained in libraries on microfiche, im-
mediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
Ack n ow led gm en t. We thank the National Insti-
tutes of Health for support of this work from grants GM-
48722 (M.N.) and CA-16954 (P.F.H.).
J O9712097