Desaturase reactions complicate the use of norcarane as a mechanistic probe. Unraveling the mixture of twenty-plus products formed in enzyme-catalyzed oxidations of norcarane

Article abstract of DOI:10.1021/jo061864r

(Chemical Equation Presented) Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that, in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPΔ2B4, CYPΔ2E1, and CYPΔ2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radical-derived product from the oxidation of norcarane, 3- hydroxymethylcyclohexene (21), and the putative cation-derived product from the oxidation of norcarane, cyclohept-3-enol (22), coelute with other oxidation products on low-polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than those previously reported.

Full text of DOI:10.1021/jo061864r

Desaturase Reactions Complicate the Use of Norcarane as a
Mechanistic Probe. Unraveling the Mixture of Twenty-Plus Products
Formed in Enzyme-Catalyzed Oxidations of Norcarane
,
†
†
†
Martin Newcomb,* R. Esala P. Chandrasena, Dharmika S. P. Lansakara-P.,
†
,‡
‡
‡
Hye-Yeong Kim, 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
0607, 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
6
men@uic.edu
ReceiVed September 8, 2006
Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations
catalyzed by several iron-containing enzymes. We report here that, in addition to oxygenated products,
norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and
3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are
produced in yields that are comparable to those of the minor products from oxidation of the norcarane.
We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four
cytochrome P450 enzymes, CYP2B1, CYP∆2B4, CYP∆2E1, and CYP∆2E1 T303A, and three diiron
enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene
monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from
Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all
reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than
20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative
radical-derived product from the oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and the
putative cation-derived product from the oxidation of norcarane, cyclohept-3-enol (22), coelute with other
oxidation products on low-polarity GC columns. The yields of product 21 found in this study are smaller
than those previously reported for the same or similar enzymes in studies where the products from norcarene
oxidations were ignored, and therefore, the limiting values for lifetimes of radical intermediates produced
in the enzyme-catalyzed oxidation reactions are shorter than those previously reported.
Introduction
validity of conclusions based on the use of probes depends on
a thorough understanding of potentially complex chemistry.
Mechanistic probes have been used for many years to reveal
details about reaction mechanisms in chemistry and biology.
The 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.
Because transient species are not followed in real time, the
existence of an intermediate can only be inferred, and the
Norcarane (1) has been applied as a probe in studies of many
1-10
enzyme-catalyzed oxidation reactions.
In most of these
(1) White, R. E.; Groves, J. T.; McClusky, G. A. Acta Biol. Med. Ger.
1979, 38, 475-489.
(2) Austin, R. N.; Chang, H. K.; Zylstra, G. J.; Groves, J. T. J. Am.
Chem. Soc. 2000, 122, 11747-11748.
(
3) Brazeau, B. J.; Austin, R. N.; Tarr, C.; Groves, J. T.; Lipscomb,
J. D. J. Am. Chem. Soc. 2001, 123, 11831-11837.
†
‡
§
⊥
(4) Auclair, K.; Hu, Z.; Little, D. M.; Ortiz de Montellano, P. R.; Groves,
J. T. J. Am. Chem. Soc. 2002, 124, 6020-6027.
University of Illinois at Chicago.
Massachusetts Institute of Technology.
Department of Pharmacology, University of Michigan.
Department of Biological Chemistry, University of Michigan.
(5) 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.
1
0.1021/jo061864r CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/18/2007
J. Org. Chem. 2007, 72, 1121-1127
1121

Newcomb et al.
works, small amounts of a putative radical-derived product and
a putative cation-derived product were detected, providing
evidence that radicals and cations were formed as intermediates,
to some extent, in the oxidation reactions. Most probes that are
used in studies of oxidizing enzymes give the same skeletal
rearrangement from radical and cation intermediates, and one
cannot conclude what type of transient was involved when
rearranged products are found. The evidence for radical-derived
products in norcarane studies and the conclusions that radical
intermediates existed is essentially unique, in disagreement with
experimental results conducted with a variety of radical clock
CHART 1
1
1
substrate probes. Moreover, results from “hypersensitive”
radical probes,1 including probes that can differentiate between
radicals and cations, indicated that no true radicals were formed
in the oxidation reactions, as discussed later. A point of concern
in most of the norcarane probe studies was that quite small
amounts of rearranged products were found, in yields compa-
rable to those of a myriad of unidentified compounds that
appeared to be derived from the norcarane substrate. Eight
oxidation products from norcarane were known, but the mixtures
appeared to contain many more products with similar molecular
weights and GC retention properties.
2
In this work, we studied oxidation reactions of norcarane by
seven iron-containing enzymes with careful attention to the
identities of minor products. In addition to oxygenated products,
norcarane was oxidized to 2-norcarene and 3-norcarene by all
of the enzymes studied, and the norcarenes were efficiently
9
oxidized by all of the enzymes. The result is that more than
2
0 primary and secondary oxidation products from norcarane
can be identified in the product mixtures. With authentic samples
of various oxidation products from 2-norcarene and 3-norcarene
the norcarenes,13 and one of those products, with the apparent
formula C7H8O2 (A), also was detected in the oxidations of
norcarane. Authentic samples of known compounds 17-22 also
1
3
available, we found that the radical- and cation-derived
rearrangement products from norcarane coelute with the nor-
carene oxidation products on low-polarity GC columns. We
conclude that the already small yields attributed to these
rearranged products probably were overestimated in earlier
studies, possibly by an order of magnitude or more.
5
,14-19
were prepared.
Enzymes. For a representative spectrum of iron-containing
enzymes, we studied the reactions catalyzed by four cytochrome
P450 enzymes (P450s) and three diiron enzymes. All of the
enzymes were purified and reconstituted for the oxidation
reactions.
Results and Discussion
The P450s studied were mammalian hepatic enzymes that
were expressed in Escherichia coli. CYP2B1 is a rat P450
Norcarane (1) and the oxidation products discussed in this
work are shown in Chart 1. 2-Norcarene (2) and 3-norcarene
2
0
induced by phenobarbital treatment. CYP∆2B4 is the rabbit
2
1
P450 induced by phenobarbital treatment; the expressed
enzyme has an 18 amino acid deletion at the N-terminal end
(
3) and products 4-16 from the oxidations of these compounds
13
are described in the accompanying paper, which reports
synthetic details for the preparations of authentic samples,
structural assignments for 4-16, and the products formed by
oxidation of the norcarenes. In this work, we used the same
numbering system for 1-16 as in the accompanying report.
Two unknown compounds were detected in the oxidations of
2
1
that does not appear to affect its function. CYP∆2E1 and
CYP∆2E1 T303A are the ethanol-inducible P450 enzyme and
its mutant, respectively, both of which also contain an 18 amino
2
2
1
3
acid deletion at the N-terminal end. This collection of P450
(
14) Chan, J. H.-H.; Rickborn, B. J. Am. Chem. Soc. 1968, 90, 6406-
411.
(15) Chini, M.; Crotti, P.; Flippin, L. A.; Gardelli, C.; Macchia, F.
6
(
6) Austin, R. N.; Buzzi, K.; Kim, E.; Zylstra, G. J.; Groves, J. T.
J. Biol. Inorg. Chem. 2003, 8, 733-740.
7) Moe, L. A.; Hu, Z. B.; Deng, D. Y.; Austin, R. N.; Groves, J. T.;
Fox, B. G. Biochemistry 2004, 43, 15688-15701.
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B. G.; Groves, J. T.; Austin, R. N. J. Inorg. Biochem. 2005, 99, 1998-
006.
9) 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.
J. Org. Chem. 1992, 57, 1713-1718.
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(
(
2
(
(19) Charette, A. B.; Francoeur, S.; Martel, J.; Wilb, N. Angew. Chem.,
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(
10) Groves, J. T. J. Inorg. Biochem. 2006, 100, 434-447.
(11) Baik, M. H.; Newcomb, M.; Friesner, R. A.; Lippard, S. J. Chem.
(20) Hanna, I. H.; Teiber, J. F.; Kokones, K. L.; Hollenberg, P. F. Arch.
Biochem. Biophys. 1998, 350, 324-332.
ReV. 2003, 103, 2385-2419.
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12) Newcomb, M.; Toy, P. H. Acc. Chem. Res. 2000, 33, 449-455.
(21) Vaz, A. D.; Pernecky, S. J.; Raner, G. M.; Coon, M. J. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 4644-4648.
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R. E. P.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg,
P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1128-1133.
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Sci. U.S.A. 1998, 95, 3555-3560.
1122 J. Org. Chem., Vol. 72, No. 4, 2007

Mixture of Products in Oxidations of Norcarane
enzymes has been studied in reactions with various mechanistic
probes.5
,21-34
The diiron enzymes studied were soluble methane monooxy-
genase (sMMO) from Methylococcus capsulatus (Bath), toluene
monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and
phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The
sMMO from M. capsulatus (Bath) has been studied with several
5
,11,27,35-37
mechanistic probes,
as has a related sMMO from
Methylosinus trichosporium OB3b.3
,11,38-41
Mechanistic studies
of the ToMO and PH enzymes from P. stutzeri OX1 have not
been reported previously, but oxidation of norcarane catalyzed
7
by a related toluene 4-monooxygenase was reported.
Desaturase Reactions of Norcarane. Small amounts of
impurities including norcarenes can be detected in distilled
samples of norcarane. In order to avoid confusion from the use
of a slightly contaminated substrate, we treated distilled nor-
carane with mCPBA to oxidize traces of olefins, and we isolated
the final sample by preparative GC. Analysis of the purified
sample by analytical GC indicated that norcarane was at least
9
3
9.96% homogeneous, and no impurities of 2-norcarene or
-norcarene could be detected to the limit of our instrumental
sensitivity. Figure 1 shows an analytical GC trace of the
norcarane used in the enzyme studies.
Following enzyme-catalyzed oxidation reactions of norcarane,
the reaction mixtures were extracted with methylene chloride,
an internal standard was added, and the product mixtures were
analyzed by GC without concentrating the samples. Both
2-norcarene and 3-norcarene were found in the product mixtures,
as illustrated in Figure 1, which shows typical GC results. The
yields of 2-norcarene and 3-norcarene, in terms of nmol of
products found, are listed in Table 1, which also includes the
total yields of other oxidation products discussed later. The
(
23) Atkinson, J. K.; Ingold, K. U. Biochemistry 1993, 32, 9209-9214.
(24) Atkinson, J. K.; Hollenberg, P. F.; Ingold, K. U.; Johnson, C. C.;
Le Tadic, M.-H.; Newcomb, M.; Putt, D. A. Biochemistry 1994, 33, 10630-
0637.
25) Newcomb, M.; Le Tadic-Biadatti, M.-H.; Chestney, D. L.; Roberts,
E. S.; Hollenberg, P. F. J. Am. Chem. Soc. 1995, 117, 12085-12091.
26) Newcomb, M.; Le Tadic, M.-H.; Putt, D. A.; Hollenberg, P. F.
J. Am. Chem. Soc. 1995, 117, 3312-3313.
27) Choi, S. Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard,
1
(
(
(
FIGURE 1. Portions of GC traces (40 °C, DB-5 column) of norcarane
before reactions and after reactions with three enzymes. The expansions
are 100× the amplitude of the lower traces. Under the GC conditions
used for these analyses, 3-norcarene and 2-norcarene elute with retention
times of 10.1 and 10.3 min, respectively. CYP ) cytochrome P450,
ToMO ) toluene monooxygenase.
S. J.; Newcomb, M.; Putt, D. A.; Upadhyaya, S. P.; Xiong, Y. J. Am. Chem.
Soc. 1996, 118, 6547-6555.
(
28) Toy, P. H.; Dhanabalasingam, B.; Newcomb, M.; Hanna, I. H.;
Hollenberg, P. F. J. Org. Chem. 1997, 62, 9114-9122.
29) Toy, P. H.; Newcomb, M.; Hollenberg, P. F. J. Am. Chem. Soc.
998, 120, 7719-7729.
30) Toy, P. H.; Newcomb, M.; Coon, M. J.; Vaz, A. D. N. J. Am. Chem.
Soc. 1998, 120, 9718-9719.
31) Newcomb, M.; Shen, R.; Choi, S. Y.; Toy, P. H.; Hollenberg,
P. F.; Vaz, A. D. N.; Coon, M. J. J. Am. Chem. Soc. 2000, 122, 2677-
686.
32) Newcomb, M.; Hollenberg, P. F.; Coon, M. J. Arch. Biochem.
Biophys. 2003, 409, 72-79.
33) Newcomb, M.; Aebisher, D.; Shen, R. N.; Chandrasena, R. E. P.;
Hollenberg, P. F.; Coon, M. J. J. Am. Chem. Soc. 2003, 125, 6064-6065.
34) Chandrasena, R. E. P.; Vatsis, K. P.; Coon, M. J.; Hollenberg,
P. F.; Newcomb, M. J. Am. Chem. Soc. 2004, 126, 115-126.
35) Liu, K. E.; Johnson, C. C.; Newcomb, M.; Lippard, S. J. J. Am.
Chem. Soc. 1993, 115, 939-947.
36) Choi, S. Y.; Eaton, P. E.; Kopp, D. A.; Lippard, S. J.; Newcomb,
M.; Shen, R. N. J. Am. Chem. Soc. 1999, 121, 12198-12199.
37) Valentine, A. M.; Le Tadic-Biadatti, M.-H.; Toy, P. H.; Newcomb,
(
1
(
norcarenes were found in high yields relative to other oxidation
products, amounting to nearly half of the total amount of
oxidation products in some cases. The initial reactions contained
ca. 1500 nmol of norcarane, and the yields of the norcarenes
relative to the initial substrate ranged from 0.1% for ToMO to
(
2
(
(
2% for CYP∆2B4. In all cases, the norcarene yields were greater
(
than the yields of any minor primary oxygenated product of
norcarane. Importantly, the norcarene yields were 1 or 3 orders
of magnitude greater than the yields of alcohols 21 and 22 (to
be discussed later), which are the “probe” rearrangement
products implicating a radical and cationic intermediate, re-
spectively.
(
(
(
M.; Lippard, S. J. J. Biol. Chem. 1999, 274, 10771-10776.
(
(
38) Jin, Y.; Lipscomb, J. D. Biochemistry 1999, 38, 6178-6186.
39) Jin, Y.; Lipscomb, J. D. Biochim. Biophys. Acta 2000, 1543, 47-
Oxidations of norcarane to give norcarenes are examples of
desaturase reactions, which are well-characterized reactions of
iron-containing enzymes. Perhaps the best known desaturase
5
9.
(
40) Liu, A. M.; Jin, Y.; Zhang, J. Y.; Brazeau, B. J.; Lipscomb, J. D.
9
enzyme is stearoyl-ACP ∆ desaturase, the soluble plant enzyme
Biochem. Biophys. Res. Commun. 2005, 338, 254-261.
(41) Wallar, B. J.; Lipscomb, J. D. Chem. ReV. 1996, 96, 2625-2657.
that oxidizes stearoyl-ACP to oleoyl-ACP, which contains a
J. Org. Chem, Vol. 72, No. 4, 2007 1123

Newcomb et al.
% norcarenese
TABLE 1. Yields of Norcarenes from Enzyme-Catalyzed Oxidations of Norcaranea
b
c
other productsd
enzyme (nmol)
2-norcarene
3-norcarene
CYP∆2E1 (1.0)
5.3
1.5
9.9
24.7
17.2
9.0
2.2
0.5
5.8
1.7
0.8
1.3
0.6
2.1
33.3
14.5
94.5
31.4
72.1
27.1
11.3
4.0
19
12
14
46
20
27
10
47
CYP∆2E1 T303A (0.5)
CYP2B1 (0.5)
CYP∆2B4 (0.5)
CYP∆2B4 (1.0)
sMMO (20)
ToMO (1.0)
PH (1.0)
0.7
1.5
a
Yields of products in nmols. b CYP ) cytochrome P450, sMMO ) soluble methane monooxygenases from M. capsulatus (Bath), ToMO ) toluene
monooxygenase from P. stutzeri OX1, PH ) phenol hydroxylase from P. stutzeri OX1. Nanomoles of enzyme used. d Yields of all other oxidation products.
Percentage of oxidation products that are norcarenes.
c
e
diiron cluster similar to that in sMMO enzymes.42 Desaturase,
or dehydrogenation, reactions have been reported for many iron-
derived alcohol 21 and cation-derived alcohol 22, SIM identi-
fication is not robust because the mass spectra of many closely
eluting products are similar. The radical-derived product 21
presents an especially serious problem because its mass spectrum
is dominated by two large fragment ions that are found in most
other mass spectra, and it elutes on a low-polarity GC column
with a retention time similar to those of products 10, 13, 16,
and A. These other products, all of which are formed in the
43
containing enzymes, including an sMMO and cytochrome
P450 enzymes.4
4,45
In comparison to the amounts of substrate norcarane in the
samples, approximately 1.5 mmol, the norcarenes represented
a minor component of the substrates, but the ratios of norcarane
to norcarenes at the end of the reactions can be misleading.
Comparisons of enzyme turnovers for oxidations of norcarane
in this work with those for oxidations of 2- and 3-norcarene
with the same enzymes13 indicate that norcarenes are more
efficiently oxidized than norcarane. For example, for sMMO,
the product yield for the oxidation of 3-norcarene1 was more
than six times as great as that for the oxidation of norcarane,
which included some products from the oxidation of the
norcarenes formed in the desaturase reactions. Another possibly
important point is that, because the norcarenes are formed in
the active site of the enzymes, they might suffer a second
oxidation in competition with escape from the active site. If a
second oxidation reaction were only 1% as fast as escape from
the active site, the amounts of the norcarene oxidation products
would exceed the amounts of the minor products from the
oxidation of norcarane, in most cases. A second oxidation of
the substrate, in competition with product release, is a concern
for the P450 enzymes, where the oxidation reaction is only
initiated when a substrate is present in the active site.
1
3
oxidations of norcarenes, contain fragment ions in their mass
spectra that match the major ions in the mass spectrum of 21.
Figure 2 shows examples of mass spectra from reaction
mixtures and from samples that illustrate the problem in
compound identification. In the mass spectral analysis of the
product mixtures, scan mode with all ion channels monitored
could not be employed for minor products (100 pmol or smaller
yields) because the background signals from materials ascribed
to the enzyme mixtures were too great. The SIM mode
effectively suppressed the background signals, but the SIM mode
with only a few ion channels cannot provide an unambiguous
identification of the products. SIM mode analysis with many
channels open gave the results in Figure 2, which are typical.
The top two spectra in Figure 2 are SIM mode mass spectra
from oxidation reactions of norcarane catalyzed by two enzymes,
where the data collection is at the appropriate elution time for
radical-derived product 21. These two mass spectra look similar,
but they do not resemble the mass spectrum of any compound
that has a similar retention time, that is, compounds 21, 16,
or A.
3
Analytical Protocol for Oxidation Products. Previous
studies did not recognize the production of norcarenes in
desaturase reactions of norcarane, and the products from the
oxidations of norcarenes were ignored in developing analytical
protocols. With an understanding that norcarene oxidation
products are formed in the enzyme-catalyzed oxidation reactions
and with the availability of authentic samples of the oxidation
products in Chart 1,13 we studied methods for identifying the
radical-derived product, 3-hydroxymethylcyclohexene (21), and
the cation-derived product, 3-cycloheptenol (22). Our results
indicate that recognition of the norcarene oxidation products is
critically important for the analyses.
Products from enzyme-catalyzed oxidations of norcarane are
formed in small amounts. Typically, the products were quanti-
fied by GC and identified by GC-mass spectrometry using
single-ion monitoring (SIM) with three to five ion channels
monitored. In regard to the probe-rearranged products, radical-
The mass spectra of 21, 16, and A collected in SIM mode
using the same channels as for the enzyme product mixtures
are shown in the three bottom panels in Figure 2. From the
mass channels 94, 95, 109, and 110, one concludes that a
mixture of compounds was present in the enzyme products. The
mixture might contain comparable amounts of 21, 16, and A,
but radical-derived product 21 clearly cannot be the only
component because the mass spectrum of 21 contains small
amounts of fragments at m/z ) 67, 68, and 95 and effectively
zero intensities at m/z ) 109 and 110.
Standard methods for GC quantification of yields also were
not robust for some of the minor norcarane oxidation products
because complete GC separation of some of the products was
not possible, and this applied to radical-derived product 21.
Authentic samples and a mixture containing radical-derived
products 3-hydroxymethylcyclohexene (21), cyclohepta-3,5-
dienol (16), and syn-2-norcaranol (17) in an approximate 1:1:
50 ratio illustrate the difficulty (Table 2). On a low-polarity,
(42) Fox, B. G.; Shanklin, J.; Somerville, C.; M u¨ nck, E. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 2486-2490.
(
(
43) Jin, Y.; Lipscomb, J. D. J. Biol. Inorg. Chem. 2001, 6, 717-725.
44) Rettie, A. E.; Rettenmeier, A. W.; Howald, W. N.; Baillie, T. A.
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Science 1987, 235, 890-893.
45) Ortiz de Montellano, P. R. Trends Pharmacol. Sci. 1989, 10, 354-
59.
21 and 16 were indistinguishable when the GC oven temperature
(
3
was at 70 °C or greater. At lower oven temperatures, the relative
1124 J. Org. Chem., Vol. 72, No. 4, 2007

Mixture of Products in Oxidations of Norcarane
FIGURE 3. GC traces (DB-5 column) for elution of a mixture of 21,
0, and 17 in a ca. 1:2:200 ratio, respectively, at varying GC column
1
oven temperatures. The retention times shown are for the 70 °C run,
and other traces were adjusted such that the peaks for 10 and 17
overlapped for each trace. The compound numbers show the positions
of the maxima for each compound.
FIGURE 2. SIM mode GC spectra obtained by monitoring 12 ion
channels. The CYP∆2B4 and sMMO spectra were measured for the
product mixture at the GC elution time where products 21 and 16 and
unknown A coelute. In each spectrum, the m/z ) 95 signal is marked
with an arrow for calibration.
FIGURE 4. Portions of GC traces (30 m × 0.32 mm DB-5 column)
of products from the oxidation of norcarane catalyzed by CYP∆2E1
T303A. Identifiable compounds are indicated with compound numbers.
Product A is an unknown compound observed in the oxidations of
norcarenes catalyzed by iron-containing enzymes.
TABLE 2. Observed Retention Times for GC Elutions as a
a
Function of Column Temperature
formed in the enzyme-catalyzed oxidations of norcarane, its
signals would overwhelm those from a small amount of 21 if
the analyses were conducted at low GC oven temperatures, thus
precluding “low temperature” quantification for 21. At higher
column temperatures, quantification of 21 remains difficult
because 21 and 16 coelute and 16 was shown to be a major
product from the oxidations of 3-norcarene by P450 and sMMO
temp (°C)
00
21
16
17
1
7.0
8.4
10.7
14.4
20.7
31.6
7.0
8.4
10.7
14.4
20.5
30.8
7.1
8.6
10.9
14.6
20.8
31.3
9
8
7
6
5
0
0
0
0
0
1
3
a
enzymes.
Retention times in minutes for elution on a 5% phenylsilicone-bonded
phase column. Compound 21 is 3-hydroxymethylcyclohexene, compound
6 is cyclohepta-3,5-dienol, and compound 17 is syn-2-norcaranol.
The differential retention time versus temperature behavior
found for the radical-derived product 21 and other alcohols with
similar GC elution times is interesting. It was also observed
for a mixture containing product 21, syn-bicyclo[4.1.0]hept-4-
en-3-ol (10), and syn-2-norcaranol (17) in a ca. 1:2:200 mixture
(21:10:17) (Figure 3). At an oven temperature of 70 °C, the
peak for radical product 21 was separated from that of 10 but
1
retention time for product 21 increased more than those for 17
and 18. Thus, 21 could be separated from 16 at a 50 or 60 °C
oven temperature, but it now overlapped with product 17 at
those temperatures. Because product 17 was the major product
J. Org. Chem, Vol. 72, No. 4, 2007 1125

Newcomb et al.
TABLE 3. Yields of Oxygenated Products from Oxidations of Norcarane Catalyzed by Iron-Containing Enzymesa
b
c
enzyme (nmol)
21+16+A
22+12
17
18+19+11
4
20
5
7
10+13
14
15
8
9
CYP∆2E1 (1.0)
0.05
0.08
0.09
0.05
0.10
0.07
0.11
0.03
0.15
0.15
0.15
0.06
0.12
0.29
0.06
0.05
7.5
5.4
24.3
7.6
33.2
7.6
17.3
9.0
5.0
0.04
0.12
1.14
0.26
0.46
0.77
0.04
0.04
0.53
0.38
4.25
1.30
3.03
1.80
0.14
0.13
0.26
0.43
0.69
0.48
0.70
0.50
0.31
0.25
0.09
0.08
0.12
0.04
0.10
d
0.18
0.09
0.26
0.16
0.41
0.06
0.03
0.03
0.09
0.09
0.18
d
0.10
0.10
0.20
0.02
0.02
0.05
0.02
0.04
d
d
d
d
d
d
d
d
d
d
d
d
CYP∆2E1 T303A (0.5)
CYP2B1 (1.0)
CYP∆2B4 (0.5)
CYP∆2B4 (1.0)
sMMO (20)
54.2
21.4
49.9
14.4
5.5
d
0.20
0.03
0.03
ToMO (1.0)
PH (1.0)
d
0.07
0.02
0.02
0.02
0.12
2.7
0.55
a
Average product yields in nmol for duplicate reactions. b CYP ) cytochrome P450, sMMO ) soluble methane monooxygenase, ToMO ) toluene
monooxygenase, PH ) phenol hydroxylase. Nanomoles of enzyme used in the oxidation reaction. d Not detected; yield less than 0.02 nmol.
c
SCHEME 1
was a shoulder on the peak from 17. At higher oven tempera-
tures, product 21 separated from 17 but overlapped with product
1
0. As a result of the variable relative retention time behavior,
the radical-derived product 21 can coelute with different
products as a function of column oven temperature, but we could
not separate it from all of the other products under any
circumstances. In order to obtain accurate yields of this product,
one needs to determine the yield of the mixture of coeluted
products and the percentage of product 21 in the mixture.
By using the mass spectra available from authentic samples,
we could identify many of the products in the mixtures formed
by the enzyme-catalyzed oxidations of norcarane. Figure 4
shows an example of a GC trace where peak areas were obtained
via flame ionization detection (FID). Peaks associated with
identifiable compounds are labeled with the appropriate com-
pound numbers. We emphasize that identification of compounds
was only possible with mass spectral analysis, which was
accomplished with a small bore capillary DB-5 column, and
we note that some peaks arise from coelution of multiple
compounds.
of the norcarenes is also novel; peaks for those products were
previously observed in GC analyses, but the compounds were
not identified.
The mechanistic probe aspects of norcarane arise because
formation of a radical or a cation at C2 results predominantly
18,47
in different fragmentation pathways
that are expected to give,
ultimately, alcohol 21 from a radical and alcohol 22 from a
cation (Scheme 1). The detection of these products provides
qualitative support for formation of the intermediates, and
because the rate constant for rearrangement of a cyclopropyl-
carbinyl radical can be estimated accurately, the percentage of
alcohol 21 formed can be used to estimate the lifetime of a
radical transient.
Unfortunately, the amounts of product 21 appear to have been
overestimated in previous studies. In Table 3, we listed the
amounts of products that elute in mixtures that might contain
alcohol 21, but inspection of the mass spectra of those peaks
shows that 21 was not the major component in the mixtures, as
illustrated in Figure 2. From these observations, we conclude
that product 21 cannot be 50% of any product mixture and might
be 10% or less. Using that range, the yields of the mixtures
that might contain radical-derived product 21, and a rate constant
Product Yields from Norcarane Oxidations. The yields of
oxygenated products from enzyme-catalyzed oxidations of
norcarane were determined by GC and GC-mass spectral
analysis of the product mixtures after concentration. The results
are listed in Table 3. We identified 18 oxygenated products for
which authentic samples were available in addition to the two
norcarenes produced in the desaturase reactions. One additional
product detected in the norcarane oxidations had the same GC
retention time and mass spectrum as an unknown product
observed in the norcarene oxidations; that product is labeled A
in Table 3 and in the accompanying paper.13 Even with the
identification of more than 20 oxidation products from norcar-
ane, the product analyses were not complete. The presence of
other, unknown oxidation products was suggested by the
complexity of the GC traces, such as that shown in Figure 4.
The total yields in nmols of oxygen-containing oxidation
8
-1 2,48
for radical ring opening of ca. 2 × 10 s ,
we can set a
limit for the lifetime of a putative radical intermediate at less
than 25 ps and possibly as small as 0.5 ps. The lower limit is
too short of a lifetime for a true radical intermediate, and it is
similar to the values found in enzyme-catalyzed reactions of
4,5
5
products for the P450 enzymes and sMMO are similar to
those reported for the same and other enzymes in comparable
studies when one adjusts for the amounts of enzymes used. The
major oxygenated products are 2-norcaranols 17 and 18, as
previously reported and as expected based on the reduced bond
dissociation energy (BDE) of approximately 3 kcal/mol for a
C-H bond adjacent to a cyclopropyl group.46 One unique aspect
of our results is that large amounts of 2-norcarene and
12,24-26,31,35-37
several hypersensitive radical probes.
Previous studies of enzyme-catalyzed oxidations of norcarane
concluded that relatively large amounts of radical-derived
product 21 were formed, and we noted the conundrum regarding
radical lifetimes estimated from those results and studies using
11
faster probe substrates. The present work appears to resolve
the conflicting conclusions. The amounts of product 21 reported
in earlier studies probably were in error due to incomplete
characterization of the minor oxidation products that overlap
with 21 in GC analyses.
3
-norcarene were found, comprising 10-47% of the oxidation
products, and these products were not previously noted. The
identification of secondary oxidation products from oxidations
(
47) Friedrich, E. C.; Jassawalla, J. D. C. Tetrahedron Lett. 1978, 19,
953-956.
(48) Newcomb, M. Tetrahedron 1993, 49, 1151-1176.
(46) Halgren, T. A.; Roberts, J. D.; Horner, J. H.; Martinez, F. N.;
Tronche, C.; Newcomb, M. J. Am. Chem. Soc. 2000, 122, 2988-2994.
1126 J. Org. Chem., Vol. 72, No. 4, 2007

Mixture of Products in Oxidations of Norcarane
2
Another possible source of error in earlier works involves
the purity of the norcarane samples employed. We found that
distilled samples of norcarane were contaminated with small
amounts of 2-norcarene, which we removed by treatment of
the distilled sample with mCPBA followed by preparative GC
collection. The introduction of impurities of norcarenes in the
norcarane sample at the outset of the study would result in even
greater yields of products that interfere with accurate quantifica-
tion of radical product 21, and the present work demonstrates
that very small amounts of norcarenes can give measurable
amounts of secondary oxidation products. The extent of
complications in earlier probe studies from contaminated
norcarane cannot be evaluated because the purity of the substrate
was not reported, and probably not determined, at the level of
parts per thousand contaminants. It is important that any future
study employing norcarane as a probe should demonstrate by
high-performance GC that the norcarane sample is not con-
taminated with norcarenes.
energy that would result from an sp -hybridized carbon atom
in a cyclopropyl ring. When the probe is capable of reacting in
a desaturase reaction, care should be taken to identify as many
products as possible, including secondary oxidation products
from the desaturated, or dehydrogenated, probe, to avoid
confusion about the identities and yields of the products.
Experimental Section
Norcarane (1) was prepared as described to the point of isolation
of the distilled product,49 which was ca. 99% pure. The mixture
was treated with mCPBA to oxidize a trace of olefinic materials
and purified by preparative GC (1/4 in. × 8 ft column, 10% SE-52
on 60/80 Chromosorb W). The isolated sample was shown to be
99.96% homogeneous by analytical GC.
The preparations of the samples of 2-norcarene (2), 3-norcarene
(3), and oxygenated products 4-16 are reported in the accompany-
ing paper.13 Literature methods were employed for the preparation
of products 17-22.5
>
,14-19
Enzyme-catalyzed oxidation reactions were conducted as
described in the accompanying paper13 using norcarane (1.5 mmol)
as the substrate. Following the reaction, an internal standard of
The desaturase reactions catalyzed by the iron-containing
enzymes complicate studies with norcarane, leading to formation
of considerable amounts of norcarenes that are subsequently
oxidized to products that interfere with the identification and
quantification of minor products from the norcarane oxidations.
The efficiency of the norcarane desaturase reactions is surpris-
ing, and one should expect that any mechanistic probe with a
reactive CH functionality adjacent to the readily oxidized
position will react readily in a desaturase reaction. Thus, any
such probe might give relatively large amounts of secondary
oxidation products that complicate mechanistic studies. Included
are the most conceivable probes, of course, but in the subset of
probe substrates containing a methyl group adjacent to a
cyclopropyl ring, the methyl group should be oxidized with
minimal desaturase activity due to the large increase in strain
2 2
1-phenyl-1-propanol was added to the product solution in CH Cl ,
and the mixture was analyzed by GC (0.32 mm × 30 m, DB-5
column) to determine the yields of 2-norcarene and 3-norcarene.
The reaction mixture was then concentrated under nitrogen, and
the concentrated sample was analyzed by GC and GC-mass
spectrometry on DB-5 columns. The results given in Table 3 are
averages of 2-4 reactions.
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: Mass spectra for com-
pounds in Chart 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
(49) Kawabata, N.; Naka, M.; Yamasita, S. J. Am. Chem. Soc. 1976,
9
8, 2676-2677.
JO061864R
J. Org. Chem, Vol. 72, No. 4, 2007 1127