Products from enzyme-catalyzed oxidations of norcarenes

Article abstract of DOI:10.1021/jo061865j

(Chemical Equation Presented) Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPΔ2E1, CYPΔ2E1 T303A, and CYPΔ2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn-and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.

Full text of DOI:10.1021/jo061865j

Products from Enzyme-Catalyzed Oxidations of Norcarenes
Martin Newcomb,*,† Dharmika S. P. Lansakara-P.,† Hye-Yeong Kim,†
R. Esala P. Chandrasena,† Stephen J. Lippard,*,‡ Laurance G. Beauvais,‡ Leslie J. Murray,‡
Viviana Izzo,‡ Paul F. Hollenberg,*,§ and Minor J. Coon*,
Department of Chemistry, UniVersity of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois
60607, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
and Departments of Pharmacology and Biological Chemistry, UniVersity of Michigan, Medical School,
Ann Arbor, Michigan 48109
men@uic.edu
ReceiVed September 8, 2006
Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-
hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary
oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as
the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.;
Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-
1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by
several enzymes were determined. Most of the products were identified by GC and GC-mass spectral
comparison to authentic samples produced independently; in some cases, stereochemical assignments
were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were
four cytochrome P450 enzymes, CYP2B1, CYP∆2E1, CYP∆2E1 T303A, and CYP∆2B4, and three diiron-
containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath),
toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from
Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are
2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn-
and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-
norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in
low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene
in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in
enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures
obtained in norcarane oxidations.
Introduction
concept of a mechanistic probe study is that a short-lived
intermediate can be revealed by a characteristic rearrangement
of a probe substrate that is observed in the reaction products.
One compound used as a probe substrate for studies of enzyme-
catalyzed oxidations is norcarane, bicyclo[4.1.0]heptane (1).
During the course of probe studies with norcarane in our
laboratories,1 we observed a large number of minor oxidation
Mechanistic probes have been used for years to reveal details
about reaction mechanisms in chemistry and biology. The
† University of Illinois at Chicago.
‡ Massachusetts Institute of Technology.
§ Department of Pharmacology, University of Michigan.
Department of Biological Chemistry, University of Michigan.
10.1021/jo061865j CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/18/2007
1128
J. Org. Chem. 2007, 72, 1128-1133

Enzyme-Catalyzed Oxidations of Norcarenes
CHART 1
SCHEME 1
SCHEME 2
The anti-epoxide 7 was previously reported to be formed by
reaction of 2-norcarene with peroxyacetic acid,5 and we prepared
compound 7 by reaction of 2-norcarene with m-chloroperoxy-
benzoic acid (mCPBA) (Scheme 2). After chromatographic
purification, compound 7 was judged to be >95% pure by NMR
spectroscopy. The structure of 7 was confirmed by 1D NOE
difference experiments that showed the enhancements in Figure
1. The chemical shift assignments for H7-endo and H7-exo were
made on the basis of the coupling constants between these two
protons and the H6 proton, which are also shown in Figure 1.
These coupling constants are similar to those for the analogous
protons in norcarane.6
When the crude product from the preparative reaction for 7
was analyzed by GC, we observed a minor product in ca. 6%
relative yield with a shorter retention time than that of 7 on a
low-polarity GC column. The mass spectrum of the minor
product was the same as that of epoxide 7. The same product
was detected in enzyme-catalyzed oxidations of 2-norcarene.
We conclude that the minor product is the syn-epoxide 6
(Scheme 2).
products that appeared to be derived from norcarane as judged
by GC retention times and mass spectra. Eventually, we realized
that, in addition to expected alcohol products, the enzymes were
oxidizing norcarane to both 2-norcarene (2) and 3-norcarene
(3) and that these alkenes were further oxidized to give small
amounts of secondary oxidation products.2 Details of the
norcarane oxidation studies are reported in the accompanying
paper.3 In this paper, we report studies of the oxidations of both
2-norcarene and 3-norcarene by several iron-containing en-
zymes. Most of the products from the oxidations of these
substrates were synthesized independently, providing firm
identifications. The results of this work are used in the
accompanying paper3 to aid in identification of the many
products from enzyme-catalyzed oxidations of norcarane.
Results and Discussion
The diastereomeric epoxides 8 and 9 were reported previ-
ously.7 These compounds were formed in comparable amounts
by the reaction of 3-norcarene with mCPBA, and they were
separated by chromatography. Reactions of the isolated epoxides
with LiNEt2 gave the known allylic alcohols 10 and 11,
respectively (Scheme 3).7
Products from Norcarene Oxidations. Iron-containing
enzymes oxidize norcarenes to give a complex mixture of
products. Chart 1 shows a collection of oxidation products that
we have characterized. Compounds 4-13 and 16 are singly
oxidized products of the norcarenes, whereas ketones 14 and
15 are doubly oxidized products. Most of the compounds in
Chart 1 were synthesized independently so that the products
from the enzyme-catalyzed oxidations could be identified by
GC and GC-mass spectral comparisons. In some cases, the
stereochemical assignments in the compounds were not certain,
and we confirmed the stereochemistry with NMR experiments.
In addition to the compounds shown in Chart 1, two other minor
products were detected in the enzyme-catalyzed oxidation
reactions of 2 and 3 but were not fully characterized.
Oxidation of 3-norcarene with chromium trioxide8 gave the
known R,â-unsaturated ketone 14 as the major product and the
previously unreported isomer 15 as a minor product (Scheme
4). The reaction of ketone 14 with DIBALH gave the allylic
alcohols 12 and 13, both new compounds, and the previously
known9 alcohol 16 (Scheme 4).
The stereochemical assignments for allylic alcohols 12 and
13 were made from coupling constant data, the results of 1D
NOE difference experiments, and the results of molecular
modeling studies. Table 1 summarizes the data. For both
isomers, Hendo and Hexo at C7 were assigned from the coupling
constants between these protons and the bridgehead proton at
C1. Molecular mechanics modeling of the two isomers indicated
that the distances between the endo and exo protons on C7 and
Ketones 4 and 5 were prepared by oxidation of the corre-
sponding alcohols (Scheme 1), as previously reported by Chan
and Rickborn.4
(1) Newcomb, M.; Shen, R. N.; Lu, Y.; Coon, M. J.; Hollenberg, P. F.;
Kopp, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 6879-6886.
(2) Newcomb, M.; Shen, R. N.; Lu, Y.; Coon, M. J.; Hollenberg, P. F.;
Kopp, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 1394.
(3) Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim,
H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg,
P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127.
(4) Chan, J. H.-H.; Rickborn, B. J. Am. Chem. Soc. 1968, 90, 6406-
6411.
(5) Lukin, K. A.; Zefirov, N. S. Zh. Org. Khim. 1988, 24, 1648-1652.
(6) Dauben, W. G.; Wipke, W. T. J. Org. Chem. 1967, 32, 2976-2979.
(7) Paquette, L. A.; Fristad, W. E.; Schuman, C. A.; Beno, M. A.;
Christoph, G. G. J. Am. Chem. Soc. 1979, 101, 4645-4655.
(8) Banwell, M. G. J. Chem. Soc., Chem. Commun. 1982, 847-848.
(9) Johnson, C. R.; Golebiowski, A.; Steensma, D. H. J. Am. Chem. Soc.
1992, 114, 9414-9418.
J. Org. Chem, Vol. 72, No. 4, 2007 1129

Newcomb et al.
TABLE 1. Data Used for Structural Assignments of Isomers 12
and 13
property or feature
12
13
HC1-HC7endo coupling constant (Hz)
HC1-HC7exo coupling constant (Hz)
HC7exo-HC7endo coupling constant (Hz)
HC2-HC7endo distance (Å)a
5.0
8.0
4.5
2.90
3.66
2.85
2.50
5.0
8.7
4.8
3.83
4.34
1.52
0
HC2-HC7exo distance (Å)a
HC7endo NOE enhancement at HC2 (%)
C7exo NOE enhancement at HC2 (%)
H
a Distance between protons in the minimum energy structure calculated
by MM2 modeling.
FIGURE 1. Selected coupling constants (top) and NOE enhancements
(bottom) for epoxide 7. The NOE values are for irradiation at HC1
and HC2
.
SCHEME 3
SCHEME 4
FIGURE 2. (A) GC retention times for compounds 4-16 on a low-
polarity column (5% phenylsilicone) at 80 °C. (B) Expansion of the
congested region of the trace in (A) with the GC trace of products
from the oxidation of 3-norcarene by CYP∆2E1 (gray line) superim-
posed. The compound numbers are shown above the lines that indicate
the retention times for each compound. Allylic alcohols 10 and 13 had
the same retention times to within 0.01 min.
the proton on C2 were shorter for isomer 12 than the respective
distances for isomer 13. A series of NOE experiments were
performed where the C7 protons on each isomer were selectively
irradiated and the absorbance enhancements for the protons on
C2 were measured. The NOE enhancements of the HC2 signal
were larger for irradiation of both of the C7 protons in isomer
12, consistent with expectations from the molecular modeling
results.
of the oxidation products are similar. For the norcarene
oxidations discussed later, we report the sum of products 10
and 13.
Although we prepared many possible oxidation products from
2- and 3-norcarene, other oxidation products were detected in
some of the enzyme-catalyzed oxidation reactions of these
substrates. One of the unidentified products appears to have a
molecular ion at m/z ) 124 (Figure 3A), which is consistent
with incorporation of two oxygen atoms and loss of four protons
from the substrate to give a highly oxidized species with the
formula C7H8O2. This product is assigned as unknown com-
pound A. It coeluted with compound 16 and was detected in
product mixtures from oxidations of both 2-norcarene and
3-norcarene. In some reactions, unknown A apparently was
formed in yields comparable to those of some of the primary
oxidation products from the norcarenes.
Another unknown oxidation product was apparent in the
reactions of 2-norcarene. Epoxide 9 should not be formed from
the oxidation of 2-norcarene, but we observed a product with a
GC retention time the same as that of epoxide 9 in the product
mixtures from most of the oxidations of 2-norcarene. This
product, labeled as unknown compound B, not only has the same
retention time as compound 9 but also has a similar mass
spectral fragmentation pattern. Figure 3B shows the fragmenta-
tion pattern for authentic epoxide 9, and Figure 3C shows the
For enzyme-catalyzed oxidation reactions of the norcarenes,
the products were quantitated by flame ionization GC and
identified by GC-mass spectral analysis. GC retention times
for compounds 4-16 are shown schematically in Figure 2A
and 2B. Figure 2B also shows a portion of a typical GC result
for a P450 enzyme-catalyzed oxidation of 3-norcarene. The GC
analyses were obtained with a low-polarity column, which gave
the best separations. Mass spectral fragmentation patterns for
these compounds are in the Supporting Information for the
accompanying paper.3
As is apparent in Figure 2, several of the oxidation products
from the norcarenes have similar GC retention times. Products
10 and 13 were not resolved under our GC conditions, and the
similar retention times for some products resulted in overlapping
peaks. In principle, the composition of merged peaks can be
determined from GC-mass spectral analysis, but this task is
not necessarily simple nor reliable because mass spectra of most
1130 J. Org. Chem., Vol. 72, No. 4, 2007

Enzyme-Catalyzed Oxidations of Norcarenes
linked to alcohol-related liver disease. In the CYP∆2E1 T303A
mutant, a highly conserved threonine located near the active
site of the enzyme was replaced with alanine; this conserved
Thr is thought to be involved in the proton delivery system in
the activation of the P450 enzymes.12 The diiron enzymes
studied were the soluble methane monooxygenase (sMMO) from
Methylococcus capsulatus (Bath), one of the more extensively
studied diiron enzymes,13-15 toluene monooxygenase (ToMO)
from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH)
from Pseudomonas stutzeri OX1. As discussed in the ac-
companying paper,3 results for norcarane oxidations by the iron-
containing enzymes studied in this work and closely related
enzymes are available.
The substrates 2-norcarene (2) and 3-norcarene (3) were
prepared by literature methods involving cyclopropanation of
1,3-cyclohexadiene and 1,4-cyclohexadiene, respectively.5,16
Purity of the substrate is an issue when quantification of small
amounts of products are necessary, and we used preparative
GC as the final step in the purification of 2 and 3. The products
used in the enzyme studies contained no detectable impurities
by NMR spectroscopy and were 99.7% homogeneous (2) and
99.3% homogeneous (3) by analytical GC.
Enzyme-catalyzed oxidations of the norcarenes involved three
or four individual experiments with each enzyme and substrate.
The norcarenes are good substrates for most of the enzymes
studied here. For the sMMO enzyme, turnover was limited,
probably because the active site is optimized for the oxidation
of methane, and 10 nmol of sMMO was used in each
experiment. The oxidation products were extracted into meth-
ylene chloride, and the product mixtures were analyzed by GC
for yield quantifications and by GC-mass spectroscopy for
identification. Table 2 contains the results. Figure 4 shows
representative GC traces for oxidations by a P450 enzyme and
two diiron enzymes.
From the total amounts of oxidation products in the final
column, 3-norcarene was determined to be a better substrate
than 2-norcarene for all of the enzymes studied. For CYP∆2B4,
five times as much product was formed from 3-norcarene as
from 2-norcarene. For the diiron enzymes, about an order of
magnitude more product was formed in the oxidations of
3-norcarene than in the oxidations of 2-norcarene.
Allylic alcohols 10-13 were the major products formed with
all of the P450 enzymes, although ketone 5 was formed in
considerable yields in the P450 oxidations of 2-norcarene. For
the diiron enzyme sMMO, allylic alcohols were the major
product from 3-norcarene, whereas ketone 5 was the major
product from 2-norcarene. Interestingly, the diiron enzymes
ToMO and PH gave high yields of epoxide 9 in oxidation
reactions of 3-norcarene but low yields of the epoxides from
2-norcarene. We noted previously that unknown product B
overlaps with epoxide 9 on the GC, but mass spectral analyses
confirmed that epoxide 9 was the major component obtained
in the oxidations of 3-norcarene.
FIGURE 3. (A) Mass spectral fragmentation pattern for unknown
product A. (B) Mass spectral fragmentation pattern for authentic product
9. (C) Mass spectral fragmentation pattern observed for unknown
product B, which eluted at the retention time expected for epoxide 9,
from the product mixture obtained in the CYP∆2E1-catalyzed oxidation
of 2-norcarene.
fragmentation pattern observed at the same retention time as
that of compound 9 from the oxidation of 2-norcarene catalyzed
by CYP∆2E1. The differences in the mass spectral fragmenta-
tion patterns are subtle. In authentic 9, the fragment with m/z
) 91 is smaller than that with m/z ) 95, and the fragment with
m/z ) 109 is greater than that with m/z ) 110. In unknown B,
the relative intensities for these two pairs are reversed. The
similarity of these fragmentation patterns illustrates the great
difficulty one has in attempting to identify a product by the use
of a few fragment peaks obtained from single-ion monitoring
(SIM) analysis of the GC trace. If SIM analysis with limited
ion channels was used for the unknown product B (Figure 3C),
one might mistakenly identify that unknown compound as
product 9.
Enzyme-Catalyzed Oxidations of Norcarenes. We studied
the oxidations of 2-norcarene and 3-norcarene with a representa-
tive spectrum of iron-containing enzymes that included four
mammalian hepatic cytochrome P450 enzymes and three diiron
enzymes. The P450 enzymes, which were expressed in Escheri-
chiacoliandpurified,wereCYP2B1,10 CYP∆2B4,11 CYP∆2E1,12
and CYP∆2E1 T303A.12 The latter three P450s contain short
deletions at the N-terminal ends of the enzymes. CYP2B1 (rat)
and CYP2B4 (rabbit) are induced in animals by phenobarbital
treatment. CYP2E1 is a constitutive and ethanol-inducible P450
The oxidation patterns observed for the norcarenes undoubt-
edly reflect substrate binding preferences in the active site
superimposed on the high reactivities expected for the double
(13) Wallar, B. J.; Lipscomb, J. D. Chem. ReV. 1996, 96, 2625-2657.
(14) Merkx, M.; Kopp, D. A.; Sazinsky, M. H.; Blazyk, J. L.; Mu¨ller,
J.; Lippard, S. J. Angew. Chem., Int. Ed. 2001, 40, 2782-2807.
(15) Baik, M. H.; Newcomb, M.; Friesner, R. A.; Lippard, S. J. Chem.
ReV. 2003, 103, 2385-2419.
(16) Gensler, W. J.; Marshall, J. P.; Langone, J. J.; Chen, J. C. J. Org.
Chem. 1977, 42, 118-125.
(10) Hanna, I. H.; Teiber, J. F.; Kokones, K. L.; Hollenberg, P. F. Arch.
Biochem. Biophys. 1998, 350, 324-332.
(11) Vaz, A. D.; Pernecky, S. J.; Raner, G. M.; Coon, M. J. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 4644-4648.
(12) Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 3555-3560.
J. Org. Chem, Vol. 72, No. 4, 2007 1131

Newcomb et al.
TABLE 2. Product Yields from Oxidations of 2-Norcarene and 3-Norcarenea
enzymeb
4
5
6
7
8
9+B
10+13
11
12
14
15
16+A
total
2-norcarene
CYP∆2E1
CYP∆2E1 T303A
CYP2B1
CYP∆2B4
sMMO
NDc
NDc
0.08
0.15
0.08
0.03
0.01
5.62
3.89
7.11
14.46
9.56
2.20
0.74
0.72
1.47
2.94
3.53
<0.08
<0.01
<0.01
0.72
1.44
3.65
5.02
<0.05
0.09
1.54
NDc
NDc
NDc
NDc
NDc
NDc
NDc
1.95
0.17
0.32
0.47
NDc
0.19
0.03
26.90
8.84
8.35
25.38
2.03
0.12
0.05
1.35
1.03
0.62
1.37
1.08
0.30
0.03
3.04
3.72
0.54
1.11
0.09
0.04
0.06
4.21
6.01
0.18
0.27
0.13
0.17
0.07
4.35
6.19
0.14
0.24
0.15
0.09
0.06
2.71
0.26
0.91
2.06
0.62
NDc
0.03
51.57
33.02
24.84
54.06
13.74
3.23
ToMO
PH
2.62
3-norcarene
CYP∆2E1
CYP∆2E1 T303A
CYP2B1
CYP∆2B4
sMMO
NDc
NDc
NDc
NDc
0.15
0.04
0.03
NDc
NDc
0.21
0.87
0.96
0.55
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
0.05
0.06
0.58
1.26
4.86
5.85
11.00
1.66
17.02
8.59
23.16
75.11
50.4
1.26
1.79
1.72
7.98
0.55
0.55
0.08
46.08
30.72
19.30
82.63
44.48
0.50
4.36
6.78
0.52
1.07
1.84
0.38
0.13
3.48
7.20
0.41
0.69
0.17
0.33
0.12
7.26
2.52
4.13
16.86
17.74
0.61
0.05
81.30
74.75
69.20
230.98
117.95
46.84
20.01
12.29
13.90
34.77
<0.15
1.03
ToMO
PH
41.61
18.86
1.19
0.20
0.39
0.09
a Product yields in nmols; averages of three or four experiments. b CYP ) cytochrome P450, sMMO ) soluble methane monooxygenase from M. capsulatus
(Bath), ToMO ) toluene monooxygenase from P. stutzeri OX1, PH ) phenol hydroxylase from P. stutzeri OX1; oxidation reactions employed 0.5 nmol of
P450 enzyme, 1.0 nmol of ToMO or PH, or 10 nmol of sMMO. c ND ) not detected.
The product distributions for norcarene oxidations found in
this work are useful for understanding and identifying the many
products found in enzyme-catalyzed oxidations of norcarane,
which result in formation of norcarenes by desaturase-type
reactions.3 One of the more important observations is that the
yields of products 16 and A were relatively large for the P450
and sMMO enzymes. Those two products coelute with the
radical-derived rearranged alcohol from the oxidations of
norcarane in GC analyses and interfere with quantification of
the latter, as discussed in the accompanying paper.3
Experimental Section
General. The preparations of 2-norcarane, 3-norcarane, and
authentic samples of oxidation products are described in the
Supporting Information. NMR spectra were recorded in CDCl3 at
300 or 500 MHz. Proton correlation was resolved with bidimen-
sional COSY experiments using the sequence cosygpqf, and
stereochemistry was determined by monodimensional NOE differ-
ence experiments. 2-Norcarene and 3-norcarene were purified by
preparative GC (10% SE-52 on 60/80 Chromosorb W, 1/4 in. × 8
ft column) at 100 °C (isothermal), and the purities of the samples
were determined by GC (DB-5, 40 °C) using flame ionization
detection. All yields and purities of other authentic samples were
determined by GC (DB-5) using flame ionization detection and by
GC-MS (HP-5MS 5% phenylmethylsiloxane column) with electron
impact (EI) ionization. Details of the sample preparations are in
the Supporting Information. Mass spectra are in the Supporting
Information for the accompanying article.3
Cytochrome P450-Catalyzed Oxidations. Cytochrome P450
2B1,10 P450 ∆2B4,11 P450 ∆2E1,12 and P450 ∆2E1 T303A12
enzymes and P450 reductase10 were expressed in E. coli and purified
as previously described. A stock solution of substrate was prepared
from substrates 2 or 3 (2 mg in 100 µL of methanol) and DLPC (2
FIGURE 4. Portions of GC traces of products from the oxidation of
2-norcarene (A) and 3-norcarene (B) with toluene monooxygenase from
P. stutzeri OX1 (ToMO), soluble methane monooxygenase from
mg) in 1.0 mL of 50 mM phosphate buffer, pH 7.4; the mixture
was sonicated and stored at 0 °C. A stock solution of NADPH was
prepared by addition of 15 mg of NADPH to 375 µL of the same
M. capsulatus (Bath) (sMMO), and cytochrome P450 2B1 (CYP2B1).
buffer, and this solution was also stored at 0 °C. Reaction mixtures
The identities of some of the products are indicated with the compound
numbers.
were prepared by mixing 0.5 nmol of enzyme, 1.0 nmol of P450
bonds and the allylic hydrogen atoms. Allylic C-H bonds have
bond dissociation energies (BDE’s) that are about 12 kcal/mol
less than those of analogous C-H bonds in saturated sys-
tems,17,18 and for the C-H bonds in 3-norcarene, the BDE will
be smaller due the adjacent cyclopropyl group.19
(17) Berkowitz, J.; Ellison, G. B.; Gutman, D. J. Phys. Chem. 1994, 98,
2744-2765.
(18) Chatgilialoglu, C.; Newcomb, M. AdV. Organomet. Chem. 1999,
44, 67-112.
(19) Halgren, T. A.; Roberts, J. D.; Horner, J. H.; Martinez, F. N.;
Tronche, C.; Newcomb, M. J. Am. Chem. Soc. 2000, 122, 2988-2994.
1132 J. Org. Chem., Vol. 72, No. 4, 2007

Enzyme-Catalyzed Oxidations of Norcarenes
reductase, and 30 µL of DLPC at 0 °C and were allowed to stand
for 10 min. Thebuffer was added to the mixture to bring the total
volume to 1.89 mL, and the mixture was allowed to stand at 0 °C.
After 10 min, 10 µL of substrate stock solution was added, and the
reaction mixture was allowed to equilibrate at 26 °C for 5 min.
The reaction was initiated by adding 100 µL of the NADPH stock
solution. Reactions were maintained at 26 °C for 30 min and then
quenched by addition of 1 mL of CH2Cl2. The organic phase was
separated, and the aqueous phase was extracted with CH2Cl2 (2 ×
1 mL). The combined organic layer was dried with MgSO4 and
filtered. An internal standard of 1-phenyl-1-propanol was added,
and the mixture was concentrated under a stream of nitrogen. The
mixture was analyzed by GC (0.32 mm × 30 m capillary column,
5% phenylsilicone-bonded phase) and GC-mass spectroscopy (0.25
mm × 30 m, capillary column, 5% phenylsilicone-bonded phase).
Soluble Methane Mmonooxygenaseonooxygenase-Catalyzed
Oxidations. The hydroxylase protein (MMOH) was purified from
M. capsulatus (Bath), as previously described.20,21 The regulatory
(MMOB) and reductase (MMOR) components were expressed
recombinantly in E. coli and purified as reported elsewhere.22,23 In
a typical reaction, a mixture of 10 nmol of MMOH, 20 nmol of
MMOB, and 5 nmol of MMOR was diluted to a volume of 400
µL with 25 mM potassium phosphate buffer (pH ) 7.0). The
enzyme was incubated for 1 min at 45 °C, and 10 µL of a solution
of substrate (2 mg of substrate in 100 µL of methanol) was added.
The oxidation was initiated by the addition of 1.6 µmol of NADH.
The mixture was gently shaken at 45 °C for 25 min and then
extracted with CH2Cl2 (3 × 2 mL). The reaction mixtures were
worked up and analyzed as described above.
protein were kindly supplied by Prof. Alberto Di Donato. The
proteins were expressed in E. coli and purified as described
elsewhere.24,25 The toluene/o-xylene monooxygenase NADH oxi-
doreductase (ToMOR) was provided by Dr. Valeria Cafaro. The
specific activity on phenol was 1.2(1) × 103 mU/mg of hydroxylase,
and the iron content for ToMOH was 4.3(2), as determined by
methods reported elsewhere.24,25 In a typical reaction, a mixture of
1 nmol of ToMOH, 66.57 nmol of ToMOB, 20 nmol of Rieske
protein, and 0.1 nmol of ToMOR was diluted to a volume of 2 mL
with 20 mM potassium phosphate buffer (pH ) 7.4). The mixture
was allowed to stand in an ice bath for 10 min. Substrate (10 µL
of a solution of 2 mg of 2-norcarene or 3-norcarene in 100 µL of
methanol) was added. The mixture was incubated at 26 °C for
10 min while shaking. The oxidation was initiated by the addition
of NADH buffer solution; the final concentration of NADH was
1 mM. The mixture was gently shaken at 26 °C for 30 min and
then extracted with CH2Cl2 (3 × 2 mL). The combined organic
phase was dried (MgSO4) and filtered. The mixtures were analyzed
by GC and GC-MS, as described previously.
Phenol Hydroxylase-Catalyzed Oxidations. The hydroxylase
component PH/H of the phenol hydroxylase complex was purified
from E. coli, strain JM109 expressing the entire ph operon cloned
into vector pGEM3Z.26 The hydroxylase moiety was purified with
a slight modification of a reported procedure.27 The iron content
was 4.2(3) per mol protein, and the specific activity on phenol was
515 (SD of 44) mU/mg of hydroxylase, determined as reported
elsewhere.24 The coupling (PHM) and reductase (PHP) components
were expressed recombinantly in E. coli, strain BL21DE3, and
purified as previously described.27 In a typical reaction, a mixture
of 1 nmol of PH/H and 2 nmol of PHM was diluted to a volume
of 2 mL with 0.1 M Tris-HCl buffer (pH ) 7.5). Substrate (10 µL
of a solution of 2 mg of 2-norcarene or 3-norcarene in 100 µL of
methanol) and 1 mM NADH were added. The mixture was
incubated at 25 °C for 5 min while shaking. The oxidation was
initiated by the addition of 2 nmol of PHP (oxidoreductase). The
mixture was gently shaken at 25 °C for 30 min and then extracted
with CH2Cl2 (3 × 2 mL). The combined organic phase was dried
(MgSO4) and filtered. The mixtures were analyzed by GC and GC-
MS, as described previously.
Toluene Monooxygenase-Catalyzed Oxidations. Plasmids con-
taining the genes for the toluene/o-xylene monooxygenase hy-
droxylase (ToMOH), the coupling protein (ToMOB), and the Rieske
(20) Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785.
(21) Beauvais, L. G.; Lippard, S. J. J. Am. Chem. Soc. 2005, 127, 7370-
7378.
(22) Coufal, D. E.; Blazyk, J. L.; Whittington, D. A.; Wu, W. W.;
Rosenzweig, A. C.; Lippard, S. J. Eur. J. Biochem. 2000, 267, 2174-2185.
(23) Kopp, D. A.; Gassner, G. T.; Blazyk, J. L.; Lippard, S. J.
Biochemistry 2001, 40, 14932-14941.
(24) Cafaro, V.; Scognamiglio, R.; Viggiani, A.; Izzo, V.; Passaro, I.;
Notomista, E.; Piaz, F. D.; Amoresano, A.; Casbarra, A.; Pucci, P.; Di
Donato, A. Eur. J. Biochem. 2002, 269, 5689-5699.
(25) Sazinsky, M.; Bard, J.; Di Donato, A.; Lippard, S. J. J. Biol. Chem.
2004, 279, 30600-30610.
Acknowledgment. This work was supported by NIH Grants
GM-48722 (M.N.), GM-32134 (S.J.L.), CA-16954 (P.F.H.), and
DK-10339 (M.J.C.).
Supporting Information Available: Synthetic details and NMR
spectra. This material is available free of charge via the Internet at
http://pubs.acs.org.
(26) Cafaro, V.; Notomista, E.; Capasso, P.; Di Donato, A. Appl. EnViron.
Microbiol. 2005, 71, 4736-4743.
(27) Cafaro, V.; Izzo, V.; Scognamiglio, R.; Notomista, E.; Capasso,
P.; Casbarra, A.; Pucci, P.; Di Donato, A. Appl. EnViron. Microbiol. 2004,
70, 2211-2219.
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