Examination of Selectivity in the Oxidation of ortho- and meta-Disubstituted Benzenes by CYP102A1 (P450 Bm3) Variants

Article abstract of DOI:10.1002/cctc.201700116

Cytochrome P450 CYP102A1 (P450 Bm3) variants were used to investigate the products arising from the P450 catalysed oxidation of a range of disubstituted benzenes. The variants used all generated increased levels of metabolites compared to the wild-type enzyme. With ortho-halotoluenes up to six different metabolites could be identified whereas the oxidation of 2-methoxytoluene generated only two aromatic oxidation products. Addition of an ethyl group markedly shifted the selectivity for oxidation to the more reactive benzylic position. Epoxidation of an alkene was also preferred to aromatic oxidation in 2-methylstyrene. Significant minor products arising from the migration of one substituent to a different position on the benzene ring were formed during certain P450-catalysed substrate turnovers. For example, 2-bromo-6-methylphenol was formed from the turnover of 2-bromotoluene and the dearomatisation product 6-ethyl-6-methylcyclohex-2,4-dienone was generated from the oxidation of 2-ethyltoluene. The RLYF/A330P variant altered the product distribution enabling the generation of certain metabolites in higher quantities. Using this variant produced 4-methyl-2-ethylphenol from 3-ethyltoluene with ≥90 % selectivity and with a biocatalytic activity suitable for scale-up of the reaction.

Full text of DOI:10.1002/cctc.201700116

Accepted Article
Title: Examination of Selectivity in the Oxidation of ortho- and meta-
Disubstituted Benzenes by CYP102A1 (P450Bm3) Variants
Authors: Samuel D Munday, Shaghayegh Dezvarei, Ian C.-K. Lau, and
Stephen Graham Bell
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Examination of Selectivity in the Oxidation of ortho- and meta-
Disubstituted Benzenes by CYP102A1 (P450Bm3) Variants
[
a]
[a]
[a]
*[a]
Samuel D. Munday, Shaghayegh Dezvarei, Ian C.-K. Lau and Dr. Stephen G. Bell
Abstract: Cytochrome P450 CYP102A1 (P450Bm3) variants were
used investigate the products arising from the P450 catalysed
oxidation of a range of disubstituted benzenes. The variants used all
generated increased levels of metabolites compared to the WT
enzyme. With ortho-halotoluenes up to six different metabolites could
be identified while the oxidation of 2-methoxytoluene generated only
two aromatic oxidation products. Addition of an ethyl group markedly
shifted the selectivity for oxidation at the more reactive benzylic
position. Epoxidation of an alkene was also preferred to aromatic
oxidation in 2-methylstyrene. Significant minor products arising from
the migration of one substituent to a different position on the benzene
ring were formed during certain P450 catalysed substrate turnovers.
For example 2-bromo-6-methylphenol was formed from the turnover
of 2-bromotoluene and the dearomatisation product 6-ethyl-6-
methylcyclohex-2,4-dienone was generated from the oxidation of 2-
ethyltoluene. The RLYF/A330P variant altered the product distribution
enabling the generation of certain metabolites in higher quantities.
Using this variant produced 4-methyl-2-ethylphenol from 3-
ethyltoluene with ≥90% selectivity and with a biocatalytic activity
suitable for scale up of the reaction.
P450BM3 (CYP102A1), a fatty acid hydroxylase, from
Bacillus megaterium, has been widely researched because of its
high catalytic activity and its ease of production and use.[5] It is a
catalytically self-sufficient enzyme in which the P450 heme
domain is fused to a reductase domain meaning it only requires
NADPH and oxygen to oxidise its substrate.[6] Many variants with
enhanced activity towards non-natural substrates and improved
product selectivity have been generated.[5, 7] As a result variants
with the ability to promote terminal and stereoselective alcohol
hydroxylation, phenol formation, and epoxidation have been
reported.[8] More recently forms which can catalyse reactions
which are not normally supported by P450 enzymes such as
cyclopropanation and amination have been designed.[ In
addition CYP102A1 and a number of evolved variants have been
shown to act on certain pharmaceuticals producing similar
metabolites to mammalian P450s.[10] Changes to oxidation
profiles can be brought about by minor alterations in substrate
structures, or by mutagenesis of the enzyme.[8b, 11] Therefore
CYP102A1 can be used to investigate of the behaviour of CYP
enzymes in the oxidative metabolism and degradation of certain
compounds.
9]
P450 catalysed oxidations of drug molecules and substrates
which contain substituted benzene, toluene and related aromatic
Introduction
[11a, 12]
moieties can often generate multiple metabolites.
These can
include products arising from rearrangement reactions.[
13]
In
Cytochrome P450 (CYP) monooxygenases catalyse the oxidation
of a wide variety of substrates using atmospheric dioxygen. Their
archetypal reaction type is the conversion of an unreactive
carbon-hydrogen bond in aliphatic and aromatic molecules to an
alcohol using a reactive iron-oxo radical cation, compound I
intermediate.[1] This ability to oxidise a broad array of organic
substrates enables CYP enzymes to have roles in many
metabolic pathways.[2] Xenobiotic detoxification and metabolite
production are two of the major roles P450s perform within
organisms.[3] One of the major roadblocks in the study of many
membrane bound P450 enzymes is that they can often be difficult
to produce and have low activities, making in depth studies of
them more complex. As such bacterial P450 enzymes have often
been used to examine the structure, function and mechanism of
this class of enzymes.[1a, b, 4]
earlier work, it was established that CYP102A1 and its variants
would preferentially oxidise most alkylbenzenes at the benzylic
position to generate an alcohol (ca. 99% for WT with
propylbenzene)
mammalian P450s.
a
similar outcome to that observed with
[8b, 14]
With toluene, o-hydroxylation was the
preferred outcome, (ca. 97% selectivity) which is a different when
compared to microsomal enzymes.[14] Subsequent work showed
that o-hydroxylation was also favoured for a range of other
monosubstituted substituted benzenes including chlorobenezene
and anisole (Fig. S1).[15] The reactions with o-, m- and p-xylene
are more complex and these are oxidised by variants of
CYP102A1 to generate multiple products (Fig. S2). p-Xylene has
been reported to be oxidised at the benzylic methyl group [16] but
others have shown that 2,5-dimethylphenol was the major
product.[17] The product profile resulting from m-xylene oxidation
consisted of 2,4-dimethylphenol as the major product (87%) with
2
,6-dimethyphenol (11%) and benzylic oxidation (2%) occurring
in lower amounts (Fig. S2).[
11a]
The product distribution obtained
[
a]
S.D. Munday, S. Dezvarei, I.C.-K. Lau & Dr. S.G. Bell
Department of Chemistry
University of Adelaide
Adelaide. SA, 5005, Australia
E-mail: Stephen.bell@adelaide.edu.au
from the oxidation of o-xylene was more diverse and consisted of
benzylic oxidation (47%) and 2,3-dimethylphenol (27%) and 3,4-
dimethylphenol (10%) formation (Fig. S2).[11a] In addition two
products arising from the shift of a methyl group were also
obtained and identified as, 2,6-dimethylphenol (8%), and 6,6-
Supporting information for this article is given via a link at the end of
the document.
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ChemCatChem
10.1002/cctc.201700116
FULL PAPER
dimethylcyclohexa-2,4-dienone (8%). This latter metabolite
resulted from dearomatisation of the benzene ring.[11a]
in `catalytically ready' conformations minimising substrate gating
and enabling more efficient substrate oxidation.[11b, 22] The R19
variant has also been demonstrated to accelerate the oxidation of
These studies determining the product distributions of
CYP102A1 oxidation of substituted benzenes can be used to
complement xenobiotic metabolism with isolated mammalian
P450 enzymes or microsomes. For example the oxidation of alkyl
substituted benzenes and styrenes by mammalian enzymes tend
to favour oxidation on the substituent whereas methoxy
substituted benzenes generate phenols as the major products.[18]
Methyl and other substituents have been reported to migrate in a
similar manner to hydrogen in the NIH shift mechanism during the
oxidation of substituted benzenes by mammalian P450s and
model systems.[19] However the reactions with microsomes or
eukaryotic P450s tend to be significantly less efficient generating
low levels of product over longer periods of time which can hinder
the identification of minor metabolites.
[
14,
non-physiological substrate but its structure is yet to be solved.
2
0]
In the CYP102A1 turnovers the partition between aromatic
and benzylic hydroxylation is thought to be primarily governed by
the orientation of the substrate with CH bond reactivity playing
an important but secondary role. In the singly substituted benzene
substrates such as toluene, and anisole the substituents must be
orientated away from the heme iron enabling aromatic
oxidation.[14-15, 17b] For substituted aromatics with ethyl and longer
alkyl chains, such as n-propylbenzene, the side chains must be
Figure 1. The location of the residues which have been mutated in the different
CYP102A1 variants (KT2, R19, RLYF/I401P and RLYF/A330P) used in the this
study. These are shown on the structure of the substrate bound (NPG: N-
palmitoylglycine) WT CYP102A1 (PDB: 1JPZ).
more closely held over the heme iron giving metabolites arising
from benzylic oxidation.[14, 20] The balance between the different
pathways in P450 catalysed oxidation appears to be delicately
poised.[
11a,
12, 16]
For example o-xylene and m-xylene show
significant differences in the relative amount of aromatic versus
benzylic oxidation products and deuteration of the methyl groups
of o-xylene led to an increase in phenolic hydroxylation by
The RLYF/A330P mutant, in which the A330P mutation is in
the active site, does not retain the ability to oxidise fatty acids.[
The structure of the A330P mutant has been solved (PDB: 3M4V)
and it shows the Pro329 residue is reoriented into the substrate
access channel, generating a smaller active site (Fig. 1).[11b] This
results in distinctive product distributions relative to the WT
enzyme and the rate accelerating variants described above. The
RLYF/A330P variant can therefore oxidise non-natural substrates
with high activity and altered product distributions.[11b]
11b]
[
21]
microsomal P450s.
Mutant variants of CYP102A1 can improve the activity of the
enzyme for alkylbenzenes and also alter the product distribution
towards or away from benzylic hydroxylation.[8b, 14] Variants of
CYP102A1 which contain the R47L and Y51F mutations (RLYF)
at the entrance of the substrate access channel have been shown
to facilitate the entry of small hydrophobic molecules.[8a, 14] Other
rate accelerating variants have been developed and these
improve the activity of the enzyme for non-natural substrates but
on the whole maintain the product selectivity of the WT
enzyme.[11b, 14, 22] Among these variants are the rate accelerators:
In this paper, we consider the in vitro oxidation of m- and o-
subsituted benzene substrates which have different substituents
on the aromatic ring. These were tested with WT CYP102A1 and
four different variants (KT2, RLYF/I401P, R19 and RLYF/A330P).
Our intention was to investigate how altering the substituent
modified the substrate-enzyme interactions and therefore the
activity and product distribution. This would enable an
assessement of the extent to which CYP102A1 can be used to
generate individual or multiple oxygenated metabolites of these
compounds and if there were any rearrangement products which
arise from the mechanism of aromatic oxidation.[
RLYF/I401P
(R47L/Y51F/I410P),
KT2
R19
The
(
(
A191T/N239H/I259V/A276T/L353I) as
R47L/Y51F/H171L/Q307H/N319Y).
well
as
22]
[
11b,
14,
17b,
20,
RLYF/I401P variant contains the I401P mutant which is on the C-
terminal side of the distal cysteine ligand while the other mutations
in the KT2 and R19 variants are remote from the active site and
1f, 19e, 25]
[
11b,
located throughout the heme domain of the enzyme (Fig. 1).
2
2a]
The crystal structures of the KT2 variant and I401P mutant,
Results
which shares characteristics in common with RLYF/I401P, have
been solved (PDB: 3PSX and PDB: 3HF2, respectively).[11b, 22b]
These reveal that both have altered conformations which in
certain aspects more closely resemble the substrate-bound WT
enzyme (PDB: 1JPZ)[23] than substrate-free CYP102A1 (PDB:
The four CYP102A1 variants; KT2, R19, RLYF/I401P and
RLYF/A330P, and the WT enzyme were tested with disubstituted
benzene substrates as well as with toluene and anisole as
controls (Fig. 2). For all the substrates tested the generic
accelerator variants enhanced the product formation activity of
BU7).[24] These rate accelerating variants are hypothesised to be
1
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ChemCatChem
10.1002/cctc.201700116
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the turnover by increasing the NADPH consumption rate and the
coupling efficiency (Table 1). KT2 was the slowest variant with
those containing the RLYF couple improving the activity by a
significantly greater degree (Tables 1). The improvement in
product formation activity over the WT enzyme ranged from 110-
fold for 2-chlorotoluene to greater than 1000-fold for 3-
bromotoluene. The acceleration of product formation activity,
to a small extent. R19 reduced the selectivity of anisole oxidation
at the para position. No demethylation of anisole was detected in
any instance. The major metabolite arising from toluene oxidation
by mammalian systems is benzyl alcohol and while aromatic
oxidation is preferred to demethylation in anisole metabolism the
para phenol is the major product.[18c, e]
–
1
-1
ranged from 240 to 1600 nmol (nmol P450) min (henceforth
-
1
abbreviated to min ), for each substrate (Table 1). In all but
one case the greatest improvement over the WT enzyme was
induced by RLYF/I401P. For example, with WT CYP102A1 no
product formation activity was observed with 3-bromo- and 3-
methoxytoluene but product formation rates of 920 and 499 min-
Table 1. In vitro oxidation activity and selectivity of P450Bm3 variants and decoy
molecules with toluene, anisole and ortho- and meta-substituted benzene
derivatives. N = NADPH turnover rate. C = coupling efficiency (%). PFR =
Product formation rate. Rates are in nmol (nmol P450) –1 min . All data are
reported as the means of at least three experiments unless otherwise indicated.
n.d. no levels of product formation were observed.
–1
1
were observed with the RLYF/I401P variant.
The product formation activities of the doubly substituted
benzenes were greater than those of toluene and anisole with
the ortho substituted substrates being converted at higher
activities than their meta substituted equivalents (Table 1). The
highest product formation rates were observed with 2-
ethyltoluene. The substrate preference of the RLYF/A330P
variant was different from the other mutants tested and the
highest activities were obtained with 2-bromo- and 2-
chlorotoluene (Table 1).
WT
KT2
R19
RLYFIP
RLYFAP
toluene
N
22  0.9
1.6  0.5
0.3  0.1
137  4
10  3
7  2
118  6
32  3
38  4
819  4
38  1
308  9
251  5
44  1
111  4
C
PFR
anisole
N
122  2
1.4  0.1
2.0  0.1
189  2
7  1
14  2
464  10
36  3
166  14
1175  16
36  2
424  33
121  2
24  0.4
29  0.3
C
PFR
2
-bromotoluene
N
61  6
2.3  0.5
1.5  0.4
265  9
46  1
123  7
1487  22
39  1
574  19
1810  7
54  2
984  39
2320  12
42  1
987  28
C
PFR
2-chlorotoluene
N
C
80  11
11  5
9  3
245  2
39  1
96  3
1034  24
42  1
435  15
1692  11
61  4
1025  70
1990  25
47  3
935  55
PFR
2-methoxytoluene
N
50  3
6.2  0.5
3.1  0.3
57  1
31  2
18  1
159  6
28  1
44  1
1090  11
63  1
689  11
606  9
52  2
315  12
C
PFR
2-ethyltoluene
N
52  4
13  5
7  3
219  9
77  1
169  8
1320  12
83  1
1100  18
1640  11
98  1
1600  23
1250  12
81  1
315  12
C
PFR
1-bromo-2-ethylbenzene
N
56  1
7  1
572  2
41  1
1370  12
43  3
2150  4
35  2
1765  71
42  1
C
PFR
3.8  0.5
237  7
599  45
840  35
737  47
2-methylstyrene
N
53 3
0.7  0.1
0.3  0.03
179  12
16  1.
28  4
313  7
24  1
75  5
754  23
32  2
240  5
325  21
5  0.3
17  0.5
C
PFR
3-bromotoluene
N
91  13
n.d.
593  12
30[
2020  34
44  1
879  21
1970  16
47  3
920  65
911  11
32  4
296  34
Figure 2. The substrates tested for in vitro activity with WT CYP102A1 and
its variants.
a]
C
n.d.
170[a]
PFR
3-methoxytoluene
N
27  11
n.d.
135  2
38  1
52  2
547  14
59  3
321  8
872  16
57  2
499  23
710  15
63  1
444  13
Most products were identified by GC coelution
experiments with authentic standards. When these were not
available analysis of the NMR or mass spectra (MS) was used
C
PFR
n.d.
3
N
-ethyltoluene
112  5
6  0.2
6.6  0.01
572  2
44  1
250  5
1320  55
50  1
656  35
1850  62
56  0.6
1030  43
1480  34
44  1
652  16
(
Supporting information). The product distributions for toluene
C
and anisole oxidation by each variant were similar to those
observed with WT. In all cases the predominant position of
hydroxylation was ortho to the substituent with a small amount
of para-hydroxylation observed (Fig. S1).[14-15, 20, 26] All the
variants, except R19, oxidised toluene at the benzylic position
PFR
[
a] determined from a single experiment.
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methyl or halide substituent. Unlike the turnover of o-xylene, no
dearomatisation product which arose via the shift of a halide to
the methyl-bound carbon or vice versa could be detected.
However the total proportion of rearrangement products with the
halotoluenes was similar to those formed with o-xylene.
Additionally there was no evidence of dehalogenation of either
substrate which has been shown to occur in the oxidation of
perhalogenated benzenes by P450s.[
27]
Scheme 1 Product distribution arising from the oxidation of 2-chlorotoluene (2-
Cl), 2-bromotoluene (2-Br) and 2-methoxytoluene (2-MeO) by the CYP102A1
variants (RLYF/A330P, WT – wild type CYP102A1 and others – the generic
accelerators, KT2, RlYF/I401P and R19).
Scheme 2 Product distribution arising from the oxidation of 2-ethyltoluene (2-
Et), 1-bromo-2-ethylbenzene (1-Br-2-Et) and 2-methylstyrene (2-MeSt) by the
CYP102A1 variants See Scheme 1 for labels. For 2-MeSt the WT and
RLYF/A330P enzymes are grouped with the others and RLYF/I401P is given
separately.
Both 2-chloro- and 2-bromo-toluene were oxidised into six
metabolites by each variant (Fig. 3 and Scheme 1). GC-MS
experiments were unable to distinguish between 4-halo-3-
methylphenol and 3-halo-4-methylphenol (X = bromo or chloro in,
4
-X-3-MP and 3-X-4-MP) and so HPLC was used to determine
Replacing the halogen atom with the larger methoxy
substituent in 2-methoxytoluene resulted in the formation of only
two products in similar quantities (Scheme 1 and Fig. S3). Both
were phenols with one metabolite 2-methoxy-3-methylphenol (2-
MeO-3-MP) identified by GC-MS coelution experiments after
synthesis of the standard (see Experimental Section). The other
was 4-methoxy-3-methylphenol (4-MeO-3-MP, Scheme 1 and
Supporting Information). The generic accelerators in RLYF/I401P,
KT2 and R19 favoured the formation of 4-MeO-3-MP, with a
maximum of 68% observed using RLYF/I401P and R19 (Scheme
the relative proportion of these products (Fig. 3). The product
distribution was relatively consistent between both substrates and
most variants However as expected the RLYF/A330P variant
generated different relative amounts of each product (Scheme 1).
For all variants other than RLYF/A330P, the major products were
the benzyl alcohols (2-BrBA and 2-ClBA, Scheme 1). A mixture of
other products resulted from hydroxylation at each position
around the benzene ring. For the rate accelerating variants the 3-
halo-2-methylphenol (3-X-2-MP) product was formed in the
second highest quantity followed by 2-halo-6-methylphenol (2-X-
1).
6-MP) and 4-halo-3-methylphenol (4-X-3-MP, Scheme 1). The
RLYF/A330P variant was less selective for benzyl alcohol
formation and 2-Cl-6-MP and 3-Br-2MP were the major products
(
Scheme 1). The 2-X-6-MP product must arise from a shift of the
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fragmentation pattern than the others (Supporting Information). o-
Fig. 3. GC-MS and HPLC analyses of o-substituted toluenes: (a) GC-MS
chromatogram of RLYF/I401P + 2-Br; (b) HPLC chromatogram of
RLYF/A330P + 2-Br (RT of 2-BrBA: 8.8 min, not shown); (c) GC-MS
chromatogram of RLYF/I401P + 2-Cl; (d) HPLC chromatogram of
RLYF/A330P + 2-Cl (RT of 2-ClBA: 13.6 min, not shown).
Xylene oxidation by CYP102A1 yields a dearomatisation product
(
metabolite has a distinctive MS spectrum compared to those
arising from phenol formation or benzylic methyl hydroxylation.
Therefore, this product was assigned as 6-ethyl-6-
6,6-DMCHD) with a short retention time (Fig. S2).[11a] This
methylcyclohexa-2,4-dienone
(6-E-6-MCHD,
Scheme
2,
Modification of the ortho substituent to an ethyl moiety
introduced a more reactive benzylic methanediyl site at the -
position. The major product of 2-ethyltoluene oxidation across all
variants was consequently shifted to oxidation at this position
generating 1-(2-methylphenyl)ethanol (1-(2-MePh)EtOH or -
ethyl-OH, Scheme 2). Further oxidation of this alcohol to give the
ketone (2-Meacetph, -ethyl-ket, 1-2%) was also observed. 2-
Ethyltoluene was oxidised into five other minor products (total
Supporting Information). The oxidation of 1-bromo-2-
ethylbenzene was also highly selective for benzylic oxidation
generating 1-(2-bromophenyl)ethanol (Scheme 2, Fig. S6 and Fig.
S7). While the selectivity for the major product was similar to that
of 2-ethyltoluene fewer minor products were observed (Fig. S6).
These were assigned as arising from further oxidation to the
ketone and phenol production (Scheme 2).
Changing the ethyl group to an alkene resulted in
<
12%) as a result of either aromatic or benzylic oxidation (Fig. S4). epoxidation being the most abundant reaction in the 2-
These were produced in small quantities and no authentic
standards were available so their full characterisation was
prohibited (Fig. S5). The mass spectra fragmentation patterns
observed for each metabolite (see Supporting Information)
indicated that three were phenolic products and another the
benzyl alcohol (2-ethylbenzyl alcohol, RT: 9.8 min). The fifth had
a significantly shorter retention time (7.0 min) and different MS
methylstyrene conversions (Fig. S7 and Experimental). A
significant amount of an aldehyde rearrangement side product
was the only other product observed (Scheme 2). More aldehyde
was observed in the turnover of the styrene by the RLYF/I401P
compared to those from the other variants.
Chiral analysis of the 1-(2-methylphenyl)ethanol, 1-(2-
bromophenyl)ethanol and 2-methylstyrene oxide products arising
from the turnovers of 2-ethyltoluene, 1-bromo-2-ethylbenzene
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and 2-methylstyrene was undertaken (Fig. S8 – S10). All the
enzyme catalysed turnovers showed little enantioselectivity with
the WT, KT2 and R19 variants and the maximum enantiomeric
excess (ee) observed being 18% (Table S1). The RLYF/I401P
variant increased the enantioselectivity of all the turnovers as did
the RLYF/A330P, however the maximum ee observed was 44%
3-bromotoluene the major metabolite from 3-methoxytoluene
oxidation also arose from hydroxylation ortho to the methyl group,
giving 4-methoxy-2-methylphenol (4-MeO-2-MP, Scheme 3).
Minor products included 2-methoxy-4-methylphenol (2-MeO-4-
MP), 2-methoxy-6-methylphenol (2-MeO-6-MP) and 2-methoxy-
5-methylphenol (2-MeO-5-MP) (Scheme
Information).
3 and Supporting
(
Table S1 and Fig. S8 -10).
The oxidation of 3-ethyltoluene by most variants generated
a mixture of two major products (Scheme 3 and Fig. S13). In
agreement with the introduction of the more reactive ethyl group
in ortho substituted benzenes, one of these was identified as 1-
(
3-methylphenyl)ethanol (1-(3-MP)EtOH; NMR in the Supporting
Information). The RLYF/A330P variant oxidised this substrate
more selectively generating 92% of the second unidentified
product (Fig. S14). This metabolite was isolated using this variant
and characterised as 4-methyl-2-ethylphenol (4-Me-2-EtP) by
matching the NMR with that reported in the literature (Supporting
Information).[28] The enantioselectivity of the 1-(3-MP)EtOH
product was determined and in most instances a small preference
for one enantiomer was observed. The highest selectivity was in
the RLYF/I401P turnover which showed an ee of 46% (Table S1
and Fig. S15).
Discussion
The product formation rates were higher for the disubstituted
benzenes than the monosubstituted toluene and anisole. The
epoxidation of 2-methylstyrene occurred at a lower activity
compared to the hydroxylation of the other substrates which
agrees with previous results comparing the oxidation of styrenes
and alkylbenzenes with CYP102A1 variants.[20] The more rigid
planar nature of the vinyl substituent may orientate the molecule
in a less favourable location for oxidation as reflected by the lower
coupling efficiencies observed despite the higher reactivity of
alkenes versus alkyl groups. The coupling efficiency and the
product formation rates were improved for variants containing the
RLYF mutations. This can be attributed to the improved uptake of
small hydrophobic substrates into the active site compared to KT2
and WT CYP102A1. In addition there may be less water in the
active site of the RLYF variants. All of the RLYF/I401P, R19 and
KT2 variants acted as generic accelerators, which improved the
activity while leaving the product distribution relatively unchanged.
RLYF/I401P altered the enantioselectivity of hydroxylation and
epoxidation suggesting the orientation of the substrate in the
active site has been changed in this variant. Due to the presence
of an active site mutation RLYF/A330P altered the selectivity of
the substrate oxidation reactions. The most dramatic example of
this being the selective formation of 4-methyl-2-ethylphenol from
3-ethyltoluene.
Scheme 3 Product distribution arising from the oxidation of 3-bromotoluene (3-
Br), 3-methoxytoluene (3-Br) and 3-ethyltoluene (3-Et) by the CYP102A1
variants. No product was observed after the turnover with the WT enzyme with
3-Br or 3-MeO. The WT CYP102A1 data is included with others for 3-Et.
Three substrates with meta arranged substituents were
analysed
(3-bromotoluene,
3-methoxytoluene
and
3-
Substituents of different size, reactivity and electronic
properties, as well as the relative positions of these groups, had
an effect on the product profiles observed. The oxidation of the
majority of the toluenes and anisoles by all the variants showed
clear preferences for hydroxylation at the CH bond next to a
methyl or methoxy group. This implied that a methyl or methoxy
methyltoluene).[
11a]
3-Bromotoluene was oxidised to four products
(
Scheme 3 and Fig. S11), with the major metabolite with all the
variants being 4-bromo-2-methylphenol (4-Br-2-MP, Scheme 3).
-Bromo-6-methylphenol (2-Br-6-MP), 2-bromo-4-methylphenol
2-Br-4-MP) and 3-bromobenzyl alcohol (3-BrBA) were formed as
minor products (Scheme 3 and Fig. S12). As with m-xylene and
2
(
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ChemCatChem
10.1002/cctc.201700116
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Scheme 4 a) Potential pathways for 2-X-6-MP formation via the ring opening of
an epoxide intermediate and shift of the methyl group. Ring opening of the
epoxide which resides between the two substituents results in two zwitterionic
intermediates. One of these intermediates is stabilised relative to the other by
resonance due to the delocalisation of the lone pair of the bromo or chloro
substituent (Scheme 4). The positive charge can reside on the electronegative
halogen, providing the stabilisation. This pathway is not stabilised by induction
but the resonance effect may provide a more significant impact on product
formation and so this pathway, which requires the shift of the methyl group
rather than the halogen, may be more likely. b) One potential route to 2-X-6-MP
via the alternative sigma bonded cation complex mechanism of Shaik.
orientation of these substrates in the active site would be
expected to be analogous to that of o-xylene with one substituent
sequestered in the active site, such that the adjacent one is
oxidised. The selectivity and improved coupling efficiency
indicated that the chloro or bromo substituents may be preferably
sequestered positioning the methyl group for benzylic oxidation.
Hydroxylation also occurred at each position around the aromatic
ring generating a wide range of metabolites in good yield
suggesting the substrate is mobile or that multiple binding
orientations are possible.
The rationalisation for the formation of the observed
products appears to be satisfied by the aromatic epoxidation
mechanism and the products which form are in line with the ring
opened intermediates that are stabilised by resonance effects
(Scheme. S1-3). The -cation complex mechanism could also be
occurring and all the products observed could be generated via
this pathway.[1g] In contrast to the o-xylene turnovers there was no
evidence of any product resulting from the shift of either
substituent to the adjacent carbon bonded to the second
substituent. The halide or methyl group could undergo a 1,2-shift
with the adjacent hydrogen to give 2-Cl-6-MP and 2-Br-6-MP
group was being sequestered in the active site such that the
adjacent aromatic CH bond was best poised for oxidation by the
enzyme.
The product distributions arising from the turnover of 2-
chlorotoluene and 2-bromotoluene were similar as were the
product formation activities and coupling efficiencies both of
which were higher than that of o-xylene. The major metabolite
generated with each of these substrates is the benzyl alcohol
(
except with the RLYF/A330P variant). This suggests that the
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ChemCatChem
10.1002/cctc.201700116
FULL PAPER
(
Scheme S1) in a similar fashion to the migration of H in the NIH
and ethyl versus t-butyl substituents may also alter the stability of
the different products and intermediates. Whether the methyl or
ethyl group or a mixture of both shift remains to be determined.
In the turnovers of 3-bromotoluene and 3-methoxytoluene
all the variants were highly selective for the formation of 4-Br-2-
MP and 4-MeO-2-MP, respectively. In both the hydroxyl group is
installed ortho to the methyl substituent (Scheme S3). The
formation of 2-MeO-5-MP requires the shift of the methoxy group
to place it para to the methyl group and no equivalent shift
occurred in the 3-bromotoluene or 3-ethyltoluene turnovers. 3-
BrBA was generated in small quantities with 3-bromotoluene. The
shift of a methoxy substituent in the turnover of 3-methoxytoluene
to generate 2-methoxy-5-methylphenol was also not observed for
2-methoxytoluene. The selectivity of the turnovers with the meta-
substituted toluenes for the major product was greater than their
ortho counterparts. All of this implies that the orientations of
significantly different substituted benzene substrates in the
enzyme active site are not the same but that molecules of similar
size and shape give rise to products which are hydroxylated at the
same relative position.
Compared to mammalian enzymes CYP102A1 oxidation of
toluene based substrates gives rise to lower benzylic oxidation
with increased hydroxylation ortho to the methyl group. In general
for both bacterial and mammalian enzyme styrene epoxidation
and benzylic oxidation of ethyl substituents bestows the major
products. Importantly methyl and halogen migrations have both
been reported previously with mammalian P450s and have now
been shown to occur with CYP102A1. The more open nature and
larger size of the active site of the mammalian enzymes
presumably favours oxidation at more reactive sites of the
substrate. The narrower active site of CYP102A1 enables the
alkylbenzene molecules to be orientated to allow oxidation at less
reactive CH bonds. The most striking example being the
oxidation of 3-ethyltoluene to 4-methyl-2-ethylphenol by the
RLYFAP mutant despite the presence of an ethyl group in the
substrate.
shift mechanism of arenes. The RLYF/A330P variant could be
used to boost the amount of products which arise from substituent
migrations. Several examples of halide or methyl group migration
have been reported during the oxidation of halobenzenes and
xylenes.[19c-e, 29] The 2-X-6-MP products could arise from the
epoxide intermediate on either side of the halide or methyl group
or from the epoxide between the two substituents (Scheme 4).
Other epoxidations could ring open to form the identical
resonance stabilised intermediate which could provide routes to
2
-X-6-MP formation. However, in this case the ring opens to form
an intermediate which is stabilised by induction only and so may
not be preferred (Scheme 4). Determining exactly which
substituent is shifted to give products such as 2-X-6-MP is not
possible from these experiments.
The oxidation of 2-methoxytoluene by the CYP102A1
variants showed significant differences to the other ortho-
substituted substrates. There was no oxidative demethylation or
benzyl alcohol formation observed suggesting sequestration of
the methyl substituent does not result in attack at the ortho
methoxy group or vice versa. In addition no phenol oxidation
product ortho to the methyl group was formed. We cannot rule out
that the positioning of this substrate in the active site is
significantly different compared to 2-ethyltoluene, o-xylene and
the halogenated toluenes. The two phenol products which were
observed, 2-MeO-3-MP and 4-MeO-3-MP, could arise from the
pathways of aromatic oxidation which pass through ring opened
intermediates that are resonance stabilised by the delocalised
negative charge introduced by the methoxy group (Scheme S2).
The major product arose from hydroxylation para to the methoxy
group which is analogous to the selectivity of anisole oxidation by
xenobiotic oxidizing P450s.
The dominant products of 2-ethyltoluene and 1-bromo-2-
ethylbenzene oxidation arose from benzylic hydroxylation of the
ethyl group. The coupling efficiency for the 2-ethyltoluene
substrate was high across each variant, indicating that the
substrate is well positioned for oxidation. The methyl group of 2-
ethylbenzene may be sequestered in the active site such that the
benzylic position in the ethyl group is placed best for hydroxylation. Conclusions
The coupling efficiency of 2-ethyltoluene oxidation was
significantly higher than those of 1-bromo-2-ethylbenzene and 2-
methylstyrene indicating a more favourable substrate binding
orientation in the CYP102A1 active site. Intriguingly 3-
ethyltoluene resulted in a mixture of two major products including
Increasing the number of substituents and their properties as well
as modifying their relative positions has a significant impact on
the regioselectivity of CYP102A1 catalysed oxidation of
substituted benzenes. The variants used all generated increased
levels of product compared to the WT enzyme with those
containing the RLYF mutations being the most successful at
4-methyl-2-ethylphenol. This indicates that the meta substituted
substrate must be positioned in the active site in different
orientations than the ortho substituted equivalent.
1
improving the product formation activity (500 to 1600 min ). This
One of the minor products of the 2-ethyltoluene was
assigned as the dearomatisation product 6-E-6-MCHD. It has
been shown that the epoxide which results in the formation of the
dearomatisation products could be expanded to generate the
oxepin, in this case, 2-E-7-MOx (Fig. S5). Oxepin formation has
been previously detected by a bacterial P450-catalysed oxidation
of tert-butylbenzene but we found no evidence for the oxepin in
this work. None was reported in the oxidation of o-xylene.[11a, 30]
The stability of the oxepin has been shown to vary depending on
the solvent and other conditions.[30] The different sterics of methyl
resulted in the generation of the metabolites in significantly higher
yields allowing facile characterisation. RLYF/A330P provided
different product distributions to the other enzymes, the most
striking example being the switch in selectivity with 3-ethyltoluene
to generate a single major product, 4-methyl-2-ethylbenzene at
1
high activity, > 650 min , despite the presence of the more
reactive benzylic CH bonds. In addition the major product from
the 2-chlorotoluene oxidation by this mutant involves a shift of one
of the substituents to generate 2-chloro-6-methylphenol. The
presence of an ortho-ethyl substituent markedly improved the
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ChemCatChem
10.1002/cctc.201700116
FULL PAPER
selectivity for oxidation at the reactive benzylic methylene.
Epoxidation of alkenes was also preferred though the activity of
this reaction was lower. The electron donating methoxy group had
a significant impact on the selectivity of oxidation. The ortho
substituted halotoluenes generated many products including a
significant proportion from the migration of one or more
substituents. The results presented here provide an insight as to
how CYP102A1 variants may be used to generate metabolites of
xenobiotic detoxification. The substituent shifts in the CYP102A1
oxidations is an important observation. The design and testing of
a wider range of substrates in which the relative position, size and
electronic properties of the substituents are varied would assist in
P450 mechanism interrogation.
NADPH turnover rate determinations
NADPH turnovers were run in 1200 μL of 50 mM oxygenated Tris, pH 7.4
at 30 °C, containing 0.2 μM enzyme and 125 μg bovine liver catalase.
Assays were held at 30 °C for 1 min prior to NADPH addition as a 20 mg
mL–1 stock to a final concentration of ~320 μM (equivalent to 2 AU). A
period of ten seconds was allowed to elapse after NADPH addition before
the absorbance decay at 340 nm was measured. Finally 1 mM substrate
added as a 100 mM stock in DMSO or EtOH. The NADPH consumption
rate was derived using ε340 = 6.22 mM−1 cm−1. All data are reported as the
mean of at least three experiments.
Product analysis
Where authentic product standards were available they were utilised for
identification purposes via HPLC and GC coelution experiments and
analysis of the MS fragmentation pattern. The epoxide product of 2-
methylstyrene was generated by adding the substrate (5 mmol) to a
solution of mCPBA (3.4 mg, 10 mmol) in acetonitrile (1 mL) and this
mixture was used for coelution experiments. 2-Methoxy-3-methylphenol
was synthesised by following a literature procedure; 2-methoxytoluene
(125 mg, 1 mmol) was added to a solution of hydrogen peroxide (30%, 2
mL) in glacial acetic acid (10 mL). The reaction was monitored by GC-MS
after adding the mixture (1 mL aliquots) into water and extraction into ethyl
acetate.[31] 1-(2-Methylphenyl)ethanol was synthesised by dropwise
addition of sodium borohydride (20 mg, 0.5 mmol) in water (5 mL) to a
stirring solution of 2'-methylacetophenone (67 mg, 0.5 mmol) in acetonitrile
(5 mL). The reactions were monitored by GC-MS.
Experimental Section
General
Reagents and organic substrates were from Sigma-Aldrich, TCI,
Fluorochem, VWR or Enamine. Buffer components, NADPH and
isopropyl--D-thiogalactopyranoside (IPTG) were from Astral Scientific,
Australia. UV/Vis spectra and spectroscopic activity assays were recorded
at 30
 0.5 °C on an Agilent CARY-60 or Varian CARY-5000
spectrophotometer. Gas Chromatography-Mass Spectrometry (GC-MS)
data were collected on a Shimadzu GC-17A using a QP5050A GC-MS
detector and a DB-5 MS fused silica column (30 m x 0.25 mm, 0.25 m).
The injector and interface were maintained at 250 °C and 280 °C,
respectively. GC and chiral analysis were performed with a Shimadzu
Tracera GC coupled to a barrier discharge ionization detector (BID)
detector using a Supelcowax column (30 m x 0.32 mm, 0.25 m) or RT-
BDEXse chiral silica column (Restek: 30 m x 0.32 mm, 0.25 μm),
respectively. In all instances helium was used as the carrier gas.
After the NADPH consumption assays or whole-cell incubations had
finished 990 μL of the reaction mixture was mixed with 10 μL of an internal
standard solution (p-cresol or trans-4-phenyl-3-buten-2-one, 20 mM stock
solution). The mixture was extracted with 400 μL of ethyl acetate and the
organic extracts were used directly for GC-MS analysis. The oven
temperature was held at 80 ºC for 3 min and then increased at 7 ºC min1
up to 220 ºC were it was held for 3 min. Metabolite yields were calculated
using calibration of authentic samples and by making the assumption that
isomeric mono-oxygenated products would give comparable responses
(Table S2). Samples containing a range of concentrations of the chosen
product including internal standard in 50 mM Tris, pH 7.4 were extracted
as above. The integrated peak areas were expressed as ratios of the
internal standard peak area and plotted against product concentration.
Cloning, expression and purification
Plasmids (pET28 based; Merck Millipore) containing the relevant
CYP102A1 gene were transformed into Escherichia coli strain BL21(DE3)
and the transformed cells were cultured in 2xYT medium at 37 C with 30
–1
[14]
μg mL kanamycin. When the OD600 of the culture reached 0.6 - 0.8 the
-
1
temperature was reduced to 20 C, 0.5 mM IPTG, 3 mL L of trace
elements solution was added (trace elements solution per litre; 0.74 g
Where HPLC analysis was required the organic solvent was removed
under a stream of nitrogen before resuspending in acetonitrile/water.
HPLC was performed using an Agilent 1260 Infinity pump equipped with
an Agilent Eclipse Plus C18 column (250 mm x 4.6 mm, 5 µm), an
autoinjector and UV detector. A gradient, 20 - 95 %, of acetonitrile (with
trifluoroacetic acid, 0.1 %) in water (TFA, 0.1 %) applied at 1 ml min was
used. Samples were identified via coelution experiments. The GC and
HPLC retention times of the substrate and products are given in the
supporting information.
CaCl
2 2
·H O, 0.18 g ZnSO
4 2
·7H O, 0.132 g MnSO
4 2
·4H O, 20.1 g Na
2
EDTA,
16.7 g FeCl
3
·6H O, 0.10 g CuSO
2
4
·5H O, 0.25 g CoCl
2
2
·6H O). After further
2
growth for 18 hours at 20 C, cells were harvested by centrifugation,
resuspended in 40 mM potassium phosphate, pH 7.4, 1 mM in dithiothreitol
1
(buffer P) and lysed by sonication on ice (forty cycles of 20 s with a minute
between each cycle). The crude extracts were then centrifuged at 37000
g for 25 min at 4 C to remove the cell debris. The supernatant was loaded
onto a GE-Healthcare DEAE fast-flow Sepharose column (200 x 50 mm)
pre-equilibrated with buffer P from which the protein was eluted using a
linear gradient of 80 - 400 mM ammonium sulphate in buffer P. The red
P450 containing fractions were collected and concentrated by ultrafiltration,
desalted using a Sephadex G-25 column pre-equilibrated with buffer P,
and re-concentrated by ultrafiltration. The solution was centrifuged at 7000
g for 10 min at 4 °C before FPLC anion-exchange purification on an GE-
Healthcare Source 15Q column (120 x 26 mm) using a linear gradient of 0
Whole-cell oxidations and product characterisation
For whole-cell oxidation turnovers the required CYP102A1 gene was
produced as described above in 200 ml of media. The cell pellet was
harvested and resuspended in 200 mL of E. coli minimal media (EMM;
K HPO , 7 g, KH PO , 3 g, Na citrate, 0.5 g, (NH ) SO , 1 g, MgSO , 0.1
2 4 2 4 3 4 2 4 4
g, 20% glucose, 20 mL and glycerol, 1% v/v per litre), each in a 2 L baffled
flask.[32] Substrate (a 1 mM aliquot) was added to each flask and shaken
-
30% 16 x phosphate buffer. Fractions containing P450 were collected,
concentrated by ultrafiltration and filter sterilised before being stored at –
0 °C in 50% (v/v) glycerol. Glycerol and salts were removed from proteins
2
(180 rpm, 30 C). Three further additions of substrate (1 mM) were made
immediately prior to experiments using a GE Healthcare 5 mL PD-10
desalting column pre-equilibrated with 50 mM Tris buffer, pH 7.4.
every 2 hours before leaving the cultures to shake overnight. During the
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ChemCatChem
10.1002/cctc.201700116
FULL PAPER
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brine (100 mL) and dried with magnesium sulphate and the organic
extracts were pooled and the solvent was removed by vacuum distillation
and then under a stream of nitrogen. The products were purified using
silica gel chromatography using a hexane/ethyl acetate stepwise gradient
ranging from 4:1 to 3:2 hexane to ethyl acetate using 5 % increases every
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S.G.B. acknowledges the ARC for
a Future Fellowship
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International Postgraduate Award (for S.D.). The authors thank
Prof. Luet-Lok Wong (University of Oxford, UK) for the gene
constructs of the CYP102A1 variants.
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[
Keywords: biocatalysis • enzyme catalysis • aromatic
hydroxylation • epoxidation • regioselective
[
[
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Entry for the Table of Contents
FULL PAPER
Cytochrome P450 CYP102A1
Samuel D. Munday, Shaghayegh
Dezvarei, Ian C.-K. Lau and Dr.
Stephen G. Bell*
(
P450Bm3) variants were used to
characterise the products arising
from the oxidation of ortho and meta
disubstituted benzenes. Highlights
include selective formation of 4-
methyl-2-ethylphenol from 3-
ethyltoluene. Products arising from
migration of substituents including
the dearomatisation product 6-ethyl-
Page No. – Page No.
Examination of Selectivity in the
Oxidation of ortho- and meta-
Disubstituted Benzenes by
CYP102A1 (P450Bm3) Variants
6
2
-methylcyclohex-2,4-dienone from
-ethyltoluene were also identified.
This article is protected by copyright. All rights reserved.