Enzyme-substrate complementarity governs access to a cationic reaction manifold in the P450BM3-catalysed oxidation of cyclopropyl fatty acids

Article abstract of DOI:10.1002/chem.201203035

The products of cytochrome P450_BM3-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.

Full text of DOI:10.1002/chem.201203035

DOI: 10.1002/chem.201203035
Enzyme–Substrate Complementarity Governs Access to a Cationic Reaction
Manifold in the P450BM3-Catalysed Oxidation of Cyclopropyl Fatty Acids
Max J. Cryle,[a, b] Patricia Y. Hayes,[a] and James J. De Voss*[a]
Abstract: The products of cytochrome
P450BM3-catalysed oxidation of cyclo-
propyl-containing dodecanoic acids are
consistent with the presence of a cati-
onic reaction intermediate, which re-
sults in efficient dehydrogenation of
the rearranged probes by the enzyme.
These results highlight the importance
of enzyme–substrate complementarity,
with a cationic intermediate occurring
only when the probes used begin to di-
verge from ideal substrates for this
enzyme. This also aids in reconciling
literature reports supporting the pres-
ence of cationic intermediates with cer-
tain cytochrome P450 enzyme/substrate
pairs.
Keywords: carbocation
·
CYP102A1 · cytochromes · enzyme
catalysis · oxidation
Introduction
One approach used extensively to characterise intermedi-
ates employs so-called cyclopropyl radical clocks. These
compounds utilise the strain inherent in a cyclopropyl ring
to report the presence of a radical a to the ring by detection
of products arising from ring opening reactions.[5] Quantifi-
cation of both the non-rearranged and ring-opened oxida-
tion products coupled with knowledge of the rate of ring
opening allows an upper limit of the lifetime for a radical in-
termediate to be calculated.
Our interest in the mechanistic aspects of fatty acid oxida-
tion by P450s led us to utilise cyclopropyl containing fatty
acids as probes of the mechanism of P450-mediated hydrox-
ylation.[6,7] These probes are closely related to the natural
substrates of the fatty acid metabolising P450 P450BM3
(CYP102A1) and revealed the presence of a radical inter-
mediate with a rate of rebound (kt) of 2–3ꢀ1010 sꢀ1.[7] This
rate is consistent for both tetradecanoate and hexadeca-
The cytochromes P450 (P450s) comprise a superfamily of
monooxygenases that play important roles in mammalian
xenobiotic detoxification and in a multitude of biosynthetic
and biodegradative pathways. They catalyse a wide variety
of oxidative transformations in Nature,[1] including the
regio- and stereoselective hydroxylation of aliphatic carbons
and, more unusually, their dehydrogenation. The mechanism
of P450 mediated hydroxylation reactions has been the sub-
ject of intensive investigation.[2] Currently, the accepted
mechanistic pathway for hydroxylation involves hydrogen
atom abstraction by a high valence iron oxo species, recently
characterised by Green and co-workers,[3] followed by “re-
bound” of the iron tethered hydroxyl radical onto the sub-
strate carbon radical to give the hydroxylated product. De-
tection of the substrate radical intermediate predicted by
this mechanism provides powerful support for this pathway.
P450 mediated dehydrogenation has been much less investi-
gated experimentally,[4] but one postulated pathway is
through radical formation, subsequent electron transfer to
form a carbocation and then loss of a hydrogen atom to gen-
erate an alkene. The structural determinants (substrate and/
or enzyme) that control partitioning between radical and
cationic pathways in P450s are not yet fully understood.
noate equivalent turnovers with P450BioI (CYP107H1) and
[6,7]
P450BM3
.
Support for a mechanism involving a radical but
not a cationic intermediate was further strengthened, as the
presence of the latter was excluded due to the observation
that cyclopropyl carbinyl cations rapidly undergo ring equili-
bration. This produces two isomeric cyclopropyl alcohols
and, through slower ring opening reactions, two isomeric ho-
moallylic alcohols.[7]
Although the results from radical clock probes have gen-
erally been consistent with the presence of a radical inter-
mediate in P450 mediated oxidation,[5] there have been indi-
cations that a cationic oxidation pathway also exists.[8] Ex-
periments performed by Groves and co-workers with the
non-haem iron containing protein toluene monooxygenase
have shown increasing amounts of cation product as the sub-
strate becomes less like the ideal one:[9] this led us to ques-
tion whether this situation may also be true for P450s.
[a] Dr. M. J. Cryle, Dr. P. Y. Hayes, Prof. Dr. J. J. De Voss
School of Chemistry and Molecular Biosciences
University of Queensland, St. Lucia Brisbane, 4072 (Australia)
Fax : (+61)7-3365-4299
E-mail: j.devoss@uq.edu.au
[b] Dr. M. J. Cryle
Current Address: Department of Biomolecular Mechanisms
Max Planck Institute for Medical Research
69120 Heidelberg (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203035.
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Table 1. Oxidation matrices observed for the oxidation of probes 1 and 2
Results and Discussion
by P450BM3 and P450BioI
.
Enzyme/
probe
Oxidation
site[a]
Hydroxylation
[%][b]
Rearranged
identity
Rearr.
[%][c]
Substrate-like C15 and C17 cyclopropyl probes had been suc-
cessfully used to demonstrate the presence of a radical and
not a cationic intermediate in both their P450BioI- and
P450BM3-catalysed oxidation. Modification of the chain
length and positioning of the cyclopropyl ring allowed the
creation of probes made iteratively less like an optimum
substrate.[10,11] Such modified probes allowed investigation
into the effect of small alterations in substrate structure
upon the oxidation pathway observed. As P450BM3 is a fast
and highly coupled enzyme for the hydroxylation of fatty
acids and is proposed to have a specific binding mode in-
volving carboxylate recognition by an arginine residue, ex-
periments focussed on this enzyme.[10,12] Furthermore, rela-
tive rates of oxidation and binding for fatty acids of differing
chain length are known,[13] allowing for rational design of cy-
clopropyl-containing fatty acid probes. The probes chosen
for these experiments were based upon dodecanoic (lauric)
acid, the shortest fatty acid found to be a good substrate for
P450BM3. The cyclopropyl ring was placed so that it occupied
the w-1 and w-2 positions in the twelve-carbon chain, with
both the (Z)-1 and (E)-2 cyclopropyl ring geometries
(Figure 1).
P450BM3/1
w-3
92ꢁ2
7
6
2.6ꢁ1.1
0.1ꢁ0.01
3.5ꢁ0.7
1.4ꢁ0.3
–
4[c]
5
P450BM3/2
P450BioI/1
w-3
w-4
w-3
97ꢁ2
–
–
2.8ꢁ0.5
95ꢁ2
–
6
4ꢁ1
4[d]
1ꢁ1
[a] The w position is the methyl terminus of the fatty acid; [b] results of
triplicate turnovers, product percent by reference to an internal standard
(see the Supporting Information); [c]ꢂ50% due to cyclopropyl cation
equilibration; [d] due to oxidation of residual 2.
These shorter chain probes showed moderate levels of
coupling (NADPH consumption: product formation, 1:
44(ꢁ5)%; 2: 43(ꢁ1)%), approximately half that seen for
the longer chain length probes with our experimental
system.[6,7] This indicated that, as expected, 1 and 2 were not
as good substrates for P450BM3 as the C15 and C17 probes.
The resultant product profile for the turnovers of 1 were far
more complicated than those observed for the C15 and C17
probes, with the formation of not only the anticipated w-3
hydroxylated product 3 but also products rationalised as oc-
curring from cationic equilibration of the cyclopropyl ring
prior to oxygen rebound (Scheme 1). Synthesis of the
E isomer of the suspected cyclopropyl alcohol product of
cationic equilibration (5) confirmed that 4 and 5 were both
present in the turnover of 1. We have previously observed
that a cation analogous to the one derived from 1 yielded al-
cohols analogous to 4 and 5. It was also possible that the 4
present was due to a small amount of the (E)-cyclopropyl
compound 2 (~1%) contaminating 1. However, analysis in-
dicated that the ratio of 1/2 was unchanged in turnovers rel-
ative to controls and therefore, more than half of 4 found in
the turnover of 1 results not from preferential oxidation of
residual 2, but rather from ring equilibration via an inter-
mediate cyclopropyl cation (Table 1). Incubation of 3 under
standard reaction conditions did not afford any rearranged
product, implying the appearance of 4 and 5 were the result
of enzymatic oxidation of 1. The product profile from the
oxidation of 2 did not reveal any rearrangement products,
continuing the trend observed with the C15 and C17 probes,
with the vast majority of product consisting of 4 (Scheme 1).
The amount of the radical rearrangement product 6 in the
incubations of 1 differed (i.e., was greatly reduced) from
that anticipated upon the basis of the results with the C15
and C17 probes (Table 1). The lack of 6 was somewhat puz-
zling as a cyclopropyl carbinyl radical was the likely precur-
sor to the cationic intermediate. Several avenues of investi-
gation were undertaken in order to explain this result. Ini-
tially, we experimentally verified the rate of rearrangement
of the secondary cyclopropyl carbinyl radical derived from
1, by generating the equivalent radical species hypothesised
to occur in the P450BM3-catalysed oxidation of 1 through
Figure 1. Cyclopropyl probes (1, 2), hydroxylated cyclopropyl (3–5) and
unsaturated compounds (6, 7) discussed in this study.
As hydroxylation of fatty acids by P450BM3 overwhelming-
ly favours oxidation at the w-1 to w-3 positions,[10] the pres-
ence of the cyclopropyl ring effectively blocks oxidation at
two of the preferred sites of oxidation. This forces oxidation
of the w-3 position, the furthest from the methyl terminus of
the fatty acid chain and thus from the haem, based upon the
[11]
known binding of fatty acids to P450BM3
.
It was anticipat-
ed that the imposition of such constraints on P450BM3 would
be important in probing the effects of a “nonsubstrate-like”
structure on the mechanism of hydroxylation. The required
probes (1, 2) and w-3 hydroxylated standards (3, 4;
Figure 1) were synthesised through comparable routes to
those already explored in the synthesis of the C15 cycloprop-
yl probe series (see the Supporting Information).[6,7] Follow-
ing the syntheses, incubation of 1 and 2 with P450BM3 was
undertaken (Table 1).
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J. J. De Voss et al.
cation (Scheme 1).[4] This com-
pound might be expected to
eliminate to give diene 7, for
which a standard was then pre-
pared. Analysis of the products
from the P450BM3 catalysed ox-
idation of 1 showed that the
diene 7 was indeed present and
at a level comparable to the
amount of 6 that would have
been expected based upon the
C15 and C17 data. This suggests
that the homoallylic radical re-
sultant from rearrangement of
the a-carbinyl radical of 1 un-
dergoes almost total conver-
sion to the diene 7. Incubation
of 6 under turnover conditions
did not afford any diene 7,
which confirmed 7, a product
of rearrangement and dehydro-
genation, as an enzyme-cata-
lysed product.
Scheme 1. Possible oxidation pathways that can be envisaged for the P450BM3-catalysed oxidation of: a) 1, and
b) 2; rearranged products indicative of a cationic intermediate are shown in red, rearranged products indica-
tive of a radical intermediate are shown in blue.
To discount the possibility
that these results were due to
probe-specific effects, the turn-
over of 1 was undertaken with
photolysis of a PTOC ester 8 (Scheme 2).[14,15] Quenching
with thiophenol led to the determination of the rates of re-
arrangement that were within 25% of the literature value
reported for (Z)-dimethyl- and (Z)-diethylcyclopropyl car-
another fatty acid metabolising P450, P450BioI (Table 1). This
enzyme has been shown to exhibit the same behaviour to-
wards C15 and C17 cyclopropyl fatty acid probes as P450BM3
,
with the detection of a radical intermediate and comparable
rate of rebound.[6,7] Fatty acid hydroxylation by P450BioI is
not the primary function of this enzyme: instead, the in-
binyl radicals (Scheme 2: 28 radical (pathway b) 6.3
108 sꢀ1, 8.0–8.6ꢀ108 sꢀ1 [16]
18 radical (pathway a) 1.8
108 sꢀ1, 1.4–2.3ꢀ108 sꢀ1[16]).[14,15]
With cationic intermediates known to be present in the
oxidation of 1 by P450BM3, it appeared possible that the ho-
moallylic radical might undergo oxidation to the homoallylic
ACHTUNGTRENNUNG
;
ACHTUNGTRENNUNG
chain cleavage of fatty acids is the biosynthetic role of
[17,18]
P450BioI
.
Results also suggest that these fatty acids are
presented as their ACP-thioesters[18] and thus a carboxylate
binding site specifically controlling the presentation of fatty
acids is unlikely to control their binding to P450BioI: rather
the binding is likely occurring in an opportunistic manner.
This is an important difference to P450BM3, where there is a
specific binding site for fatty acids that more tightly controls
the access of fatty acids to the active site.[11] The oxidation
of 1 by P450BioI (Table 1) did not reveal any cationic rear-
rangement products (detection of 4 is explained by the re-
sidual amount of 2 (~1%) present in 1) and yielded a rate
of rebound for 1 of approximately 2ꢀ1010 sꢀ1. This rate is in
agreement with the rates determined with the use of longer
chain (Z)-cyclopropyl probes and indicated that the results
of oxidation of 1 by P450BM3 were caused by the interaction
of the probe with the enzyme, not merely a chemical proper-
ty of the probe itself.
Conclusion
Scheme 2. Timing of the rate of ring opening of the a-carbinyl radical ex-
pected for compound 1, by irradiation of 8 and quenching with thiophe-
In conclusion, the turnover of the cyclopropyl containing
nol.
fatty acid probes 1 and 2 by P450BM3 has revealed a compli-
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P450BM3-Catalysed Oxidation of Cyclopropyl Fatty Acids
FULL PAPER
(E,E)-7, 7.52 min; syn-5, 11.65 min; syn-4, 11.75 min; anti-4, 11.85 min;
(E)-6, 11.96 min; anti-5, 12.18 min; anti-3, 12.30 min; syn-3, 12.67 min.
Relative MS response of standards: single ion monitoring, electron
impact, 70 eV. Quantification performed through comparison to an inter-
nal standard (phenyl acetic acid). Relative responses of 3/4 (m/z 158)/5
(m/z 155)/6 (m/z 166)/7 (m/z 224), 100:65:140:2.
cated product profile for 1 that provides evidence for a cati-
onic intermediate. The initial formation of a radical product
appears to be confirmed by the formation of small amounts
of ring opened homoallylic alcohol and moderate levels of
diene, presumably resulting from electron transfer from an
intermediate homoallylic radical.[19] These results suggest
that cationic pathways can play a role in some P450-mediat-
ed oxidations depending upon the complementarity of the
substrate and the enzyme active site. This may enable recon-
ciliation of literature reports supporting the presence of cati-
onic intermediates with certain enzyme/substrate pairs. It
has recently been proposed that a fatty acid P450 complex
can exist in reactive and non-reactive conformations follow-
ing formation of the reactive iron oxo species.[20] Such a
paradigm might be expanded to suggest that different sub-
strates could react from different conformations, governed
by enzyme–substrate complementarity, and this could lead
to electron transfer competing with oxygen rebound and the
appearance of cationic intermediates. Our results also sup-
port the intermediacy of a cation in P450-mediated dehydro-
genations and demonstrate that this reaction competes with
hydroxylation without obvious structural or electronic alter-
ation to the enzyme or substrate.
Radical kinetic studies: Two Pyrex tubes (1 cmꢀ6 cm) were fitted with
stirring bars, septa and purged with nitrogen. A solution of thiophenol in
degassed THF (2.51m, 1.0 mL, final concentration 2.50m) was added to
each tube and the tubes protected from light. The tubes were cooled to
ꢀ788C and a solution of 8 in degassed THF (16.0 mm, 5.0 mL, 80.0 nmol)
was added to each. The solutions were allowed to equilibrate at room
temperature for 2 min before being irradiated by a 150 W tungsten fila-
ment lamp at room temperature for a period of 4 h. This experiment was
also repeated in duplicate with a tenfold increase in the number of moles
of 8. The reaction mixtures were analysed by GC-MS to determine the
products present and the ratio of products produced: VF-23 ms column;
splitless mode; column flow 2.7 mLminꢀ1; total flow 46.4 mLminꢀ1; injec-
tor 2508C; detector 2508C; oven 1008C (1.0 min equilibration) hold for
4.0 min, ramp 168Cminꢀ1 to 2508C and hold for 10.0 min (total program
time 21.0 min). Retention times: (E)-10, 5.58 min; (Z)-10, 5.63 min; Me-
2, 5.90 min; Me-1, 6.38 min; (E)-9, 6.00 min; (Z)-9, 6.18 min.
Calculation of rate of rebound producing a secondary radical:[15] Using
Equation (1) and the ratio of Me-1 and 9 [determined by GC-MS as
8.0:10.0
108 sꢀ1
G
E
.
Calculation of rate of rebound producing a primary radical:[15] Using
Equation (1) and the ratio of Me-1 and 10 [determined by GC-MS as
3.0:1.0
108 sꢀ1
N
E
Experimental Section
Kr ¼ kH½PhSHꢃ½9 or 10ꢃ=½Me-1ꢃ
ð1Þ
½15ꢃ
where kH ¼ 2:0 ꢄ 108 sꢀ1
,
and ½PhSHꢃ ¼ 2:50 m:
Enzymatic turnover of substrates 1 and 2 catalysed by P450BM3: Turn-
overs of 1 and 2 were performed by using the following protocol.
P450BM3 (final concentration 2 mm), substrate (final concentration 500 mm)
and catalase (final concentration 1 mm) were combined in an Eppendorf
tube (1.5 mL) in phosphate buffer (100 mm, pH 7.4). NADPH (final con-
centration 500 mm) was added to the solution (such that the final volume
of solution was 500 mL) and this was incubated for 30 min at 378C. Phe-
nylacetic acid (final concentration 125 mm) was then added as an internal
standard. The reaction was then loaded onto solid phase extraction
(SPE) cartridges (Phenomenex, Strata-X reverse phase absorbant,
30 mgmLꢀ1 capacity) that had been washed with methanol (1 mL) and
equilibrated with distilled water (1 mL). The samples were washed with
distilled water (0.5 mL) before being dried under vacuum for 30 min. The
samples were then eluted with ethyl acetate (0.5 mL) and treated with di-
azomethane. The samples were concentrated before being analysed by
GC-MS. Quantification of absolute amount of product and therefore the
coupling of the substrate was performed by comparison to the internal
standard referring to a standard curve (vide infra). Turnovers were per-
formed in triplicate for each substrate, with two controls also performed,
one with the exclusion of P450BM3 and the other with the exclusion of
NADPH. Incubations of 3 and 6 under the turnover conditions indicated
above with the exclusion of NADPH were also performed to determine
the stability of the cyclopropyl alcohol and homoallylic alcohol products
under the assay conditions.
Compound characterisation for 1–10
9-(Z)-2-Methylcyclopropyl)-nonanoic acid (1): Colourless oil; 1H NMR
(400 MHz, CDCl3): d=ꢀ0.38 (1H, m), 0.50–0.72 (3H, m), 0.98 (3H, d,
J1 =6.0 Hz), 1.15–1.40 (12H, m), 1.61 (2H, quint, J1 =7.2 Hz), 2.32 (2H,
t, J1 =7.6 Hz), 10.8 ppm (1H, brs); 13C NMR (100 MHz, CDCl3): d=9.3,
12.0, 13.2, 15.7, 24.7, 28.4, 29.0, 29.2, 29.45, 29.53, 30.1, 34.1, 180.5 ppm;
elemental analysis calcd (%) for C13H24O2: C 73.54, H 11.39; found: C
73.75, H 11.31; GC-MS (methyl ester) (%): 194 (3.0), 152 (6.9), 123 (6.5),
110 (12.1), 96 (17.6), 74 (42.6), 55 (69.3), 41 (100).
9-(E)-2-Methylcyclopropyl)-nonanoic acid (2): Colourless oil; 1H NMR
(400 MHz, CDCl3): d=0.11 (2H, m), 0.35 (2H, m), 0.98 (3H, t, J1 =
6.2 Hz), 1.16 (2H, quint, J1 =6.8 Hz), 1.20–1.45 (10H, m), 1.63 (2H,
quint, J1 =7.4 Hz), 2.32 (2H, t, J1 =7.5 Hz), 10.9 ppm (1H, brs);
13C NMR (100 MHz, CDCl3): d=9.3, 12.6, 12.9, 19.0, 19.9, 24.7, 29.0,
29.2, 29.4, 29.6, 34.1, 34.2, 180.5 ppm; elemental analysis calcd (%) for
C13H24O2: C 73.54, H 11.39; found: C 73.66, H 11.10; GC-MS (methyl
ester) (%): 194 (3.9), 152 (8.2), 123 (7.8), 110 (14.4), 96 (22.5), 74 (46.9),
55 (81.9), 41 (100).
Methyl (Z)-9-(2-methylcyclopropyl)-9-hydroxynonanoate (3): Standard
synthesised as the methyl ester; colourless oil; 1H NMR (400 MHz,
CDCl3): d=ꢀ0.26 (1H, dd, J1 =5.3, 10.0 Hz), 0.52 (1H, dt, J1 =4.5,
8.4 Hz), 0.60–0.74 (2H, m), 1.01 (3H, d, J1 =6.0 Hz), 1.17–1.27 (8H, m),
1.30–1.69 (5H, m), 2.18 (2H, t, J1 =7.4 Hz), 3.13 (1H, td, J1 =6.1, 9.1 Hz),
3.43 ppm (3H, s); 13C NMR (100 MHz, CDCl3): d=9.2, 10.9, 13.4, 22.8,
24.9, 25.7, 29.0, 29.1, 29.5, 34.0, 37.7, 51.4, 72.9, 174.3 ppm; GC-MS (%):
200 (0.2), 158 (13.5), 143 (3.4), 115 (11.4), 101 (8.6), 98 (9.4), 87 (39.9), 85
(40.6), 74 (26.9), 67 (36.2), 57 (43.2), 55 (64.3), 43 (100), 41 (89.0); ele-
mental analysis calcd (%) for C14H26O3: C 69.38, H 10.81; found: C 69.08,
H 10.99.
Enzymatic turnover of 1 with P450BioI: Turnover of 1 was performed by
using the above protocol, with the following enzymes included instead of
P450BM3: P450BioI (final concentration 5 mm), cindoxin (final concentration
10 mm) and E. coli flavodoxin reductase (final concentration 1 mm).
GCMS turnover analysis conditions: GC Program for analysis of the
methyl esters formed after turnover of 1 and 2 and subsequent diazome-
thane treatment plus methyl ester standards on VF-23 ms column: split-
less mode; column flow 2.5 mLminꢀ1; total flow 46.4 mLminꢀ1; injector
2508C; detector 2508C; oven 1008C (1.0 min equilibration) hold for
2.0 min, ramp 168Cminꢀ1 to 1808C and hold for 5.0 min, ramp
168Cminꢀ1 to 2508C and hold for 14.0 min (total program time
30.38 min). Retention times (methyl esters): 2, 5.95 min; 1, 6.44 min;
Methyl (E)-9-(2-methylcyclopropyl)-9-hydroxynonanoate (4): Standard
synthesised as the methyl ester; colourless oil; 1H NMR (400 MHz,
CDCl3, mixture of 2 isomers): d=0.21–0.27 (2H, m), 0.35 (1H, td, J1 =
4.7, 8.4 Hz), 0.42 (1H, td, J1 =4.9, 8.4 Hz), 0.54–0.65 (3H, m), 0.65–0.72
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J. J. De Voss et al.
(1H, m), 1.03 (3H, d, J1 =5.8 Hz), 1.05 (3H, d, J1 =5.9 Hz), 1.28–1.33
(16H, m), 1.51–1.65 (10H, m), 2.30 (4H, dt, J1 =1.4, 7.6 Hz), 2.85–2.92
(2H, m), 3.66 ppm (6H, s); 13C NMR (100 MHz, CDCl3): d (mixture of 2
isomers)=10.6, 10.8, 11.1, 11.2, 18.4, 18.7, 24.9, 25.57, 25.62, 26.7, 27.0,
29.0, 29.18, 29.21, 29.5, 34.0, 37.1, 37.4, 51.4, 76.4, 76.5, 174.3 ppm; GC-
MS (%): 200 (0.1), 158 (14.2), 143 (4.1), 115 (11.4), 101 (8.4), 98 (11.0),
87 (41.9), 85 (44.3), 74 (28.5), 67 (37.8), 57 (43.8), 55 (66.6), 43 (100), 41
(84.8); elemental analysis calcd (%) for C14H26O3: C 69.38, H 10.81;
found: C 69.39, H 10.74.
Methyl 10-methyldodec-9-enoate (10): Colourless oil; 1H NMR
(500 MHz, CDCl3): d=0.91 (5.10H, d, J1 =6.6 Hz), 0.93 (0.90H, d, J1 =
6.2 Hz), 1.20–1.35 (8H, m), 1.60 (2H, quint, J1 =7.5 Hz), 1.92 (0.3H, q,
J1 =6.6 Hz), 1.97 (1.7H, q, J1 =6.6 Hz), 2.27 (2H, t, J1 =7.5 Hz), 2.57
(1H, 2 octets, J1 =6.6 Hz), 3.64 (3H, s), 5.18 (1.85H, m), 5.32 ppm
(0.15H, m); 13C NMR (125 MHz, CDCl3, Z isomer only): d=22.7, 23.2,
24.9, 26.4, 27.2, 29.0, 29.1, 29.8, 34.1, 51.4, 127.4, 137.5, 174.3 ppm; GC-
MS (%): 226 [M+ ] (1.1), 194 (3.7), 152 (2.5), 139 (9.2), 74 (18.9), 69
C
(68.7), 55 (94.4), 41 (100); elemental analysis calcd (%) for C14H26O2: C
74.29, H 11.58; found: C 73.99, H 11.76.
Methyl 8-(E)-2-(1-hydroxyethyl)cyclopropyl)octanoate (5): Standard syn-
thesised as the methyl ester; colourless oil; 1H NMR (400 MHz, CDCl3,
mixture of 2 isomers): d=0.11 (2H, ddd, J1 =4.7, 8.4, 12.7 Hz), 0.20 (1H,
td, J1 =5.0, 8.3 Hz), 0.28 (1H, dt, J1 =4.7, 9.0 Hz), 0.45–0.6 (4H, m), 0.91–
1.35 (22H, m), 1.17 (3H, d, J1 =6.2 Hz), 1.19 (3H, d, J1 =6.2 Hz), 1.51–
1.60 (4H, m), 2.11 (4H, t, J1 =7.4 Hz), 2.92–2.99 (2H, m), 3.56 ppm (6H,
s); 13C NMR (100 MHz, C6D6): d (mixture of 2 isomers)=9.5 and 10.2,
22.9 and 23.0, 25.21 and 25.24, 27.3 and 27.4, 29.37 and 29.41, 29.54,
29.57, 29.61, 29.63, 29.8 and 29.9, 34.1, 50.9, 71.6, 173.4 ppm; GC-MS
For complete synthetic details see Supporting Information.
Acknowledgements
(%): 227 [M+ ꢀCH3] (0.2), 187 (1.8), 155 (25.4), 135 (5.6), 119 (18.3), 111
C
The authors would like to acknowledge financial support from the Aus-
tralian Research Council Grant DP110104455 (J.D.V.) and from the
Deutsche Forschungsgemeinschaft through the Emmy Noether Program
(M.J.C.).
(9.6), 98 (13.7), 87 (13.9), 83 (20.6), 74 (19.5), 69 (24.5), 67 (29.5), 55
(100), 45 (35.9), 43 (97.7), 41 (73.9); elemental analysis calcd (%) for
C14H26O3: C 69.38, H 10.81; found: C 69.18, H 10.91.
Methyl 12-hydroxytridec-9-enoate (6): Standard synthesised as the methyl
1
ester; colourless oil; H NMR (400 MHz, CDCl3): d=1.16 (2.25H, d, J1 =
[1] F. P. Guengerich, Curr. Drug Metab. 2001, 2, 93; M. J. Cryle, J. E.
Stok, J. J. De Voss, Austr. J. Chem. 2003, 56, 749.
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Montellano, J. J. De Voss, Nat. Prod. Rep. 2002, 19, 477; S. Shaik, S.
Cohen, Y. Wang, H. Chen, D. Kumar, W. Thiel, Chem. Rev. 2010,
110, 949.
6.0 Hz), 1.18 (0.75H, d, J1 =6.0 Hz), 1.20–1.40 (8H, m), 1.59 (2H, quint,
J1 =7.0 Hz), 1.86 (1H, brs), 2.00 (2H, quint, J1 =7.0 Hz), 2.05–2.20 (2H,
2 m), 2.27 (2H, t, J1 =7.4 Hz), 3.64 (3H, s), 3.70–3.85 (1H, m), 5.37 (1H,
m), 5.49 ppm (1H, m); 13C NMR (100 MHz, CDCl3, E isomer only): d=
22.6, 24.9, 28.9, 29.0, 29.1, 29.3, 32.6, 34.1, 42.5, 51.4, 67.2, 125.8, 134.6,
174.3 ppm; GC-MS (%): 224 [M+ꢀH2O] (0.4), 198 (2.4), 166 (7.9), 124
(11.7), 96 (19.5), 84 (23.2), 74 (40.8), 59 (18.5), 55 (62.5), 45 (100), 43
(49.1), 41 (59.0); elemental analysis calcd (%) for C14H26O3: C 69.38, H
10.81; found: C 69.37, H 11.04.
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W. J. Ehlhardt, P. Kulanthaivel, D. L. Lanza, C. A. Reilly, G. S. Yost,
J. Pharmacol. Exp. Ther. 2007, 322, 843; C. J. C. Whitehouse, S. G.
Bell, L.-L. Wong, Chem. Eur. J. 2008, 14, 10905.
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109, 3415; P. R. Ortiz de Montellano, S. D. Nelson, Arch. Biochem.
Biophys. 2011, 507, 95.
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2005, 70, 2455.
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Chem. Commun. 2004, 512.
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A. D. N. Vaz, M. J. Coon, J. Am. Chem. Soc. 2000, 122, 2677; M.
Newcomb, R. Shen, Y. Lu, M. J. Coon, P. F. Hollenberg, D. A. Kopp,
S. J. Lippard, J. Am. Chem. Soc. 2002, 124, 6879; P. H. Toy, M. New-
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J. A. Peterson, Arch. Biochem. Biophys. 1992, 292, 20.
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7160; C. J. C. Whitehouse, S. G. Bell, L.-L. Wong, Chem. Soc. Rev.
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Clarkson, W. E. Smith, M. R. Cheesman, A. W. Munro, J. Biol.
Chem. 2004, 279, 23274.
Methyl tridec-9E,11-dienoate (7): Standard synthesised as the methyl
ester; colourless oil; 1H NMR (400 MHz, CDCl3): d=1.20–1.40 (8H, m),
1.58 (2H, m), 1.71 (2H, quint, J1 =10.0 Hz, J2 =1.6 Hz), 2.01 (1.16H, q,
J1 =7.0 Hz), 2.07 (0.84H, q, J1 =7.0 Hz), 2.27 (2H, t, J1 =7.2 Hz), 3.64
(3H, s), 5.34 (0.58H, d of q, J1 =10.8 Hz, J2 =7.0 Hz), 5.51 (0.84H, m),
5.63 (0.58H, d of t, J1 =15.0 Hz, J2 =7.5 Hz), 5.97 (1.42H, m), 6.29 ppm
(0.58H, d of d of q, J1 =15.0 Hz, J2 =10.8 Hz, J3 =1.2 Hz); 13C NMR
(100 MHz, CDCl3): d=13.2, 18.0, 24.9, 28.93, 28.98, 29.05, 29.08, 29.3,
29.7, 32.5, 32.8, 34.1, 51.4, 123.9, 125.4, 126.7, 129.5, 130.3, 131.7, 132.0,
134.4, 174.3 ppm; GC-MS (%): 224 [M+ ] (10.7), 192 (2.3), 150 (8.4), 95
C
(23.7), 81 (73.0), 68 (93.7), 67 (71.6), 41 (100); elemental analysis calcd
(%) for C14H24O2: C 74.95, H 10.78; found: C 74.67, H 10.94.
2-(2-Methyl-Z-cyclopropyl)-decanedioic acid, 10-methyl ester, 1-pyridine-
1
2-thione-N-oxycarbonyl ester (8): Yellow oil; H NMR (500 MHz, CDCl3,
Z isomer only): d=0.15 (1H, q, J1 =5.0 Hz), 0.79 (1H, m), 0.99 (2H, m),
1.03 (3H, d, J1 =6.0 Hz), 1.20–1.35 (7H, m), 1.49 (1H, m), 1.60 (2H,
quint, J1 =7.0 Hz), 1.70 (1H, m), 1.96 (1H, m), 2.27 (1H, m), 2.28 (2H, t,
J1 =7.5 Hz), 3.63 (3H, s), 7.67 (1H, t of d, J1 =7.0 Hz, J2 =1.9 Hz), 7.16
(1H, d of d of d, J1 =9.0 Hz, J2 =7.0 Hz, J3 =1.9 Hz), 7.48 (1H, d of d,
J1 =7.0 Hz, J2 =1.3 Hz), 7.67 ppm (1H, d of d of d, J1 =9.0 Hz, J2 =
1.9 Hz, J3 =0.6 Hz); 13C NMR (125 MHz, CDCl3, Z isomer only): d=
10.5, 11.5, 13.3, 18.6, 24.9, 27.2, 28.99, 29.02, 29.3, 32.7, 34.1, 43.1, 51.4,
112.5, 133.3, 137.5, 137.7, 171.8, 174.3, 176.0 ppm; GC-MS (%): 347 [M+
C
ꢀMeOH] (0.3), 335 (3.3), 302 (11.19), 138 (100), 136 (65.7), 112 (75.2),
111 (77.7), 78 (45.9), 67 (57.3), 41 (51.2); due to the light sensitivity of 8,
high resolution mass spectral data could not be obtained.
Methyl tridec-9-enoate (9): Colourless oil; 1H NMR (500 MHz, CDCl3):
d=0.86 (0.75H, t, J1 =7.5 Hz), 0.87 (2.25H, t, J1 =7.5 Hz), 1.25–1.40
(10H, m), 1.59 (2H, quint, J1 =7.3 Hz), 1.90–2.05 (4H, m), 2.28 (2H, t,
J1 =7.5 Hz), 3.64 (3H, s), 5.29–5.37 ppm (2H, m); 13C NMR (125 MHz,
CDCl3, E isomer only): d=13.8, 22.9, 24.9, 27.1, 29.05, 29.10, 29.12, 29.3,
[14] D. H. R. Barton, D. Crich, W. B. Motherwell, Tetrahedron 1985, 41,
3901.
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111, 268.
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5687.
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M. J. Cryle, J. J. De Voss, Chem. Commun. 2004, 86.
29.6, 34.1, 51.4, 129.7, 129.9, 174.3 ppm; GC-MS (%): 226 [M+ ] (0.5), 194
C
(2.8), 152 (4.3), 110 (7.8), 74 (32.6), 67 (24.0), 55 (88.3), 41 (100); elemen-
tal analysis calcd (%) for C14H26O2: C 74.29, H 11.58; found: C 74.27, H
11.71.
15998
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Chem. Eur. J. 2012, 18, 15994 – 15999

P450BM3-Catalysed Oxidation of Cyclopropyl Fatty Acids
FULL PAPER
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M. J. Cryle, I. Schlichting, Proc. Natl. Acad. Sci. USA 2008, 105,
15696.
tion manifold makes the proposed ionic elimination mechanism the
simpler alternative.
[20] Z. Su, X. Chen, J. H. Horner, M. Newcomb, Chem. Eur. J. 2012, 18,
[19] It is possible that dehydrogenation to yield 7 occurs by H atom
transfer from the intermediate homoallylic radical rather than via a
cation. However, the demonstrated presence of cations in the reac-
2472.
Received: August 27, 2012
Published online: October 29, 2012
Chem. Eur. J. 2012, 18, 15994 – 15999
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
15999