Regioselective o-hydroxylation of monosubstituted benzenes by P450 BM3

Article abstract of DOI:10.1002/anie.201303986

Dream come true: A new monooxygenase catalyst shows excellent activity for the hydroxylation of halogenated benzenes, anisole, and toluene with almost complete ortho regioselectivity (see scheme; R=F, Cl, Br, I, CH₃, OCH₃). The substrates were hydroxylated at room temperature in water without cosolvent using molecular oxygen as oxidant. Copyright

Full text of DOI:10.1002/anie.201303986

Angewandte
Chemie
DOI: 10.1002/anie.201303986
Aromatic Hydroxylation
Regioselective o-Hydroxylation of Monosubstituted Benzenes by P450
BM3**
Alexander Dennig, Nina Lꢀlsdorf, Haifeng Liu, and Ulrich Schwaneberg*
Halogenated phenols are widely used building blocks in the
synthesis of vitamins, lipid-lowering agents, and other drugs.[1]
Commonly, halogenated phenols are produced in chemo-
synthetic processes[2] requiring energy-intensive downstream
processing steps and hazardous chemicals, for example (Hg/
Ti) acetate derivatives for the synthesis of iodophenol.[2b,3]
Chloro- and bromophenols are produced by electrophilic
halogenation reactions employing Br2 and Cl2, yielding
mixtures of o- and p-phenol derivatives as well as additional
by-products (e.g. dichlorophenol).[1b,c] Consequently, purifi-
cation of isomeric products is required for drug synthesis.[1e,4]
Guaiacol and its derivatives vanillin and eugenol are inter-
mediates in the perfume and flavor industry and common
antioxidants in the treatment of cancer, cardiovascular
disorders, and Parkinsonꢀs and Alzheimerꢀs diseases.[5] Guaia-
col can be isolated from plants or synthesized from catechol
by reaction with stoichiometric amounts of NaOH in the
presence of other corrosive reagents.[6] The direct chemical
hydroxylation of substituted benzenes is unattractive since
oxygenation reactions on nonactivated C atoms are unselec-
tive[7] and do not occur on the aromatic ring.[8]
mechanism in which the hydroxylated substrate forms an
epoxide intermediate followed by NIH shift (1,2-hydride
shift) to retain the phenol was reported by de Vissier and
Shaik[15] and experimentally proven by Whitehouse et al.[14]
Recently, the P450 BM3 variant M2 (R47S/Y51W/I401M)
was engineered in our group and found to catalyzed the
efficient and regioselective aromatic hydroxylation of p-
xylene.[12a]
Herein we report the aromatic hydroxylation of six
monosubstituted benzenes (Scheme 1) with the engineered
variant M2 to study the production of o-substituted phenols
by direct hydroxylation with oxygen at room temperature in
water (KPi 50 mm, pH 7.5). In a systematical approach the
Biocatalysis offers alternative and attractive routes to the
direct aromatic hydroxylation of benzenes.[4,9] When water
can serve as the reaction medium the use of organic solvents is
minimized and toxic or corrosive by-products such as HBr
and HCl can be avoided.[1a,b,7,9a,10] Especially the potential of
regio- and enantioselective catalysis makes direct enzymatic
hydroxylations attractive for the production of pharmaceuti-
cals.[4,7,9b] A major limitation for the application of biocata-
lysts in technical processes is their often insufficient opera-
tional stability.[11] Only a few peroxidases are known to
halogenate phenols with moderate selectivity.[9a] In this
regard, protein engineering offers powerful methods to
tailor enzymes to meet specific industrial demands.[12]
P450 BM3, a heme-dependent and industrially important
monooxygenase,[13] catalyzes the direct aromatic hydroxyl-
Scheme 1. A) Aromatic substrates of the P450 BM3 variant M2 (R47S/
Y51W/I401M). a) P450 BM3 M2 (0.075 mm), O2, RT, potassium phos-
phate buffer (50 mm, pH 7.5), NADPH. B) Products obtained after
aromatic hydroxylation with P450 BM3 M2. C) Selected drugs that
contain structure motifs in (B) (http://www.drugbank.ca). FDA-
approved drugs are marked with an asterisk.
ation of benzenes with high selectivity and activity.[12a,d,14]
A
[*] A. Dennig, N. Lꢀlsdorf, Dr. H. Liu, Prof. Dr. U. Schwaneberg
Institute of Biotechnology, RWTH Aachen University
Worringerweg 1, 52074 Aachen (Germany)
E-mail: u.schwaneberg@biotec.rwth-aachen.de
Homepage: http://www.biotec.rwth-aachen.de/
influence of ring substituents (F, Cl, Br, I, CH3, and OCH3) on
regioselective hydroxylation was investigated. A list of 25
further compounds investigated including nitrobenzene and
aniline can be found in the Supporting Information (Table S1)
along with a protocol for the semi-preparative-scale produc-
tion of guaiacol and o-cresol. The highly sensitive, regiose-
lective 4-AAP assay was used to quantify the products[16] and
GC-FID and GC-MS were employed with commercial stand-
[**] This research was supported financially by the EU-funded 7th
Framework Project OXYGREEN (FP7-KBBE; project reference:
212281).
Supporting information for this article (including the protocol for
semi-preparative-scale production of o-phenol compounds, GC-FID
chromatograms, and GC-MS data for all obtained products)is
available on the WWW under http://dx.doi.org/10.1002/anie.
201303986.
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Table 1: Catalytic performance of P450 BM3 wild-type and variant M2 for the aromatic hydroxylation of six substrates.
iTOF[a]
[sÀ1
TOF[b]
U/mgP450
C [%][d]
Phenol [%]
m-/p-
[c]
Variant
Substrate
]
[300 sÀ1
]
o-
WT
M2
n.d.
38.6Æ1.8
362.3Æ91.9
n.d.
19.5
11Æ4
44Æ4
>90
>95
n.d./<10
n.d./<5
anisole
2427.4Æ172.7
WT
M2
n.d.
14.6Æ0.6
83.5Æ8.4
n.d.
7.4
10Æ3
48Æ4
>95
>99
<1[e]
n.d.
toluene
1062.9Æ107.6
WT
M2
n.d.
n.d.
n.d.
n.d.
0.02
0
n.d.
49
n.d.
n.d./51
fluorobenzene
chlorobenzene
bromobenzene
iodobenzene
67.2Æ30.7[g]
6Æ3
WT
M2
n.d.
12.3Æ1.3
237.5Æ38.6
n.d.
6.2
6Æ3
>96
>99
<4
<1
1923.4Æ116.5
28Æ4
WT
M2
n.d.
7.7Æ1.3
169.8Æ12.3
1993.7Æ88.3
n.d.
3.8
5Æ3
>97
>99
<3
n.d./<1
29Æ6
WT
M2
n.d.
2Æ0.7
n.d.
205.4Æ20.8
n.d.
0.97
0
n.d.
>99
n.d.
23Æ8
n.d.[f]
[a] Initial turnover frequency (iTOF) [mmolproduct mmolP450À1 sÀ1]. [b] TOF [mmolproduct mmolP450À1 300sÀ1]. [c] mmolphenol minÀ1 mgP450À1. [d] Coupling
efficiency. [e] 3% yield of benzyl alcohol. [f] 51.1% yield of phenol (GC area; see also Figure S5 in the Supporting Information); values determined after
300 s are partially limited by NADPH. [g] Determined after 30 min reaction time. n.d.=not detected.
ards to determine the regioselectivity of the reaction (Sup-
porting Information). Notably, aromatic hydroxylations were
performed most efficiently in aqueous solution without
additional cosolvents that could interfere in the purification
of the products, despite the poor solubility of the substrates in
water (< 2 gLÀ1).[17]
control and yield a mixture of ortho and para products.[20] The
importance of amino acid residue F87 for the aromatic
hydroxylation of p-xylene was reported previously by us and
attributed to strong p–p interactions(Figures 1 and 2).[12a,21]
The regioselective hydroxylation of substituted benzenes by
wild-type BM3 and the M2 variant is controlled through space
by the binding and orientation of benzene substrates in
a defined manner towards T268, a key residue in dioxygen
activation and substrate recognition (Figure 1).[13,22] Small
compounds such as benzenes have to be positioned correctly
in the active site for regiospecific hydroxylation since the
large substrate-binding pocket of P450 BM3 can harbor, for
instance, polycyclic aromatic hydrocarbons.[23] P450 BM3
controls reactions through space, and the intrinsic reactivity
of substrates does not play a dominant role during catalysis in
Table 1 and Figure S1 in the Supporting Information show
that wild-type P450 BM3 catalyzes product formation with
moderate activities which do not exceed 0.6 U/mgP450 (guaia-
col). The wild-type enzyme does not convert fluoro- and
iodobenzene at a detectable level. Variant M2 could effi-
ciently hydroxylate all six substrates including fluoro- and
iodobenzene. Based on initial turnover frequency (iTOF)
values, the aromatic hydroxylation of anisole was performed
with particular efficiency with 19.5 U/mgP450. An iTOF of
38.6 sÀ1 and specific activity of 19.5 U/mgP450 is, to the best of
our knowledge, the highest reported rate of product forma-
tion for a monooxygenase-catalyzed direct aromatic hydrox-
ylation of a benzene compound.[13] Interestingly, the produc-
tivity for the formation of guaiacol is about three times higher
than for o-cresol (7.4 U/mgP450), likely due to differences in
steric demands (Table 1, Scheme 1).[12a,13] Toluene and
bromo- and chlorobenzene are also converted much faster
by P450 BM3 M2 than by the wild-type enzyme (12.7-fold, 8-
fold, and 11.7-fold, respectively) even under NADPH-limited
conditions (TOF values in Table 1 and Figure S1).
The wild-type and M2 enzymes have excellent regiose-
lectivity (> 90% o-hydroxylation) and M2 reaches almost
quantitative values for the o-hydroxylations of toluene and
halogenated substrates. The only exception is fluorobenzene
(Table 1) which is converted with a ratio of 49:51 (ratio of o-
to p-hydroxylation); the m-hydroxylation that was reported
for eukaryotic P450s is not observed.[18] The fluoro substituent
is highly electronegative[19] and can potentially reduce p–p
interactions with phenylalanine87 (F87). This decrease in the
p–p interactions could result in the loss of precise steric
Figure 1. Active site of P450 BM3 (PDB: 1BU7) with the docked
anisole substrate (ball-and-stick representation) in T-shape orientation
and highest interaction energies (A: À4.98 kcalmolÀ1, B: À5.46 kcal
molÀ1).[29] Conformation A suggests hydroxylation at the ortho position;
conformation B suggests hydroxylation preferentially at the para posi-
tion. The heme center and the residues T268 (dioxygen activation)[22]
and F87 (indispensable for aromatic hydroxylation)[12a] are depicted in
stick representation. The distance (in ꢁ) between the C atoms of
anisole closest to the water ligand bound to the heme iron are
indicated with arrows.
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contrast to most chemical catalysts.[24] Figure 2 shows the
substrate docked into an active site cavity located between
the key catalytic residues F87 and T268 and the heme iron.
The substrate fits excellently in a T-shape orientation with
strong binding energies of À4.98 and À5.46 kcalmolÀ1.
tivity values. The main differences between the substituents
(OCH3, CH3, Cl, Br, I, F) are steric demand and electro-
negativity.[19] The p–p interactions between residue F87 and
monosubstituted benzenes can occur in four orientations:
a) p–p stacked, b) dimer, c) T-shaped, and d) inverse T-
shaped.[21] Depending on the binding state the distance
between two aromatic rings can vary from 3.5 (b) to 6.0 ꢁ
(d) leading to interaction energies ranging from À5.38 (a) to
À0.88 kcalmolÀ1 (d) for benzene and hexafluorobenzene
dimers.[21] The high regioselectivity of BM3 and variant M2
is a first indication that the selected benzene substrates
(Table 1) have a strongly preferred orientation for o-hydrox-
ylation.
The docking of anisole into the P450 BM3 active site
yielded only the T-shape orientation relative to F87 (A and B
in Figure 1) with binding energies of À4.98 (A) and
À5.46 kcalmolÀ1 (B). The two orientations (inversion of the
OCH3 group) with the highest binding energies are shown in
Figure 2, suggesting an interaction with F87 in a T-shape
orientation. Conformation A would direct the ortho position
directly towards the reactive oxygen species which is located
between T268 and the heme center.[14,15] Conformation B
would preferentially yield p- and m-hydroxylated phenols,
which were produced in minor amounts (Table 1). The latter
results indicate that conformation A (Figure 1) is the pre-
ferred orientation mode for BM3 wild-type and M2. Despite
the good agreement between the docking studies and the
experimental data, the docking studies neglect the dynamics
within the binding pocket and heme during the hydroxylation
reactions.[13] The following correlation between atom size,
electronegativity, and reactivity of the halogenated substitu-
ents was experimentally observed: M2 activity: Cl > Br> I
(Table 1).[14] Fast chlorobenzene hydroxylation could be
attributed to an electron-withdrawing effect which could
promote faster formation of the epoxide intermediate.[15]
Electron-rich substituents such as iodine tend to stabilize
the epoxide intermediate, therefore re-aromatization could
be slower according to the proposed mechanism.[30] In
addition, hydroxylation by P450 BM3 M2 leads to dehaloge-
nation of iodobenzene to phenol (51.1%, GC).[31]
In summary, we have reported the first direct hydroxyl-
ation of halogenated benzenes with nearly perfect regiose-
lectivity for chloro-, bromo-, and iodobenzene by an engi-
neered P450 BM3 variant (M2) and the first P450-catalyzed
hydroxylation of iodobenzene. All phenols were produced at
room temperature in water without co-solvent and with
molecular oxygen. The reported phenols are important
synthons and direct hydroxylation offers novel options for
the synthesis of hydroxylated halobenzenes. Notably, the
engineered M2 variant has excellent selectivity (> 99%) and
for a P450 enzyme an excellent activity (e.g. anisole hydrox-
ylation: 19.5 U/mgP450; 0.67 gLÀ1 product; TTN of 6195).
Figure 2. Active-site cavity P450 BM3 (PDB: 1BU7) with anisole
docked in two T-shaped binding orientations. The red surface indicates
the space filling of residues in the P450 BM3 active site at a distance
of less than 4 ꢁ to anisole. Distances are given in ꢁ between anisole
and the key residues F87 and T268 as well as heme iron. Both
orientations would lead preferentially to the formation of o-phenols.
Docking was achieved using the VINA docking plug-in[32] for
YASARA.[29]
An important parameter for describing the performance
of P450 monooxygenases is the coupling efficiency, a measure
of the efficient use of NADPH.[13] High coupling efficiencies
require that substrates are positioned in specific orientations
so that an efficient transfer of the activated oxygen can be
achieved from BM3 to the targeted C atom.[25] The BM3 wild-
type showed coupling efficiencies ranging from 5 to 11%
(Table 1). No NADPH consumption was observed for iodo-
benzene, suggesting that it is not a substrate for BM3 wild-
type. One reason could be, as reported for DMSO, an
interaction of the substrate with amino acid residue R47
which restricts the entrance to the active site.[26] In the case of
fluorobenzene, NADPH was consumed without being
hydroxylated, indicating that the substrate is bound within
the active site inducing electron transfer to the heme iron (low
spin to high spin)[27] which enables oxygen binding.[28] Oxygen
is finally reduced to hydrogen peroxide or another reactive
oxygen species when the bound substrate is “loosely fit-
ting”[13] and no hydrogen atom is positioned for abstrac-
tion.[25a] Compared to BM3 wild-type, variant M2 displays
significantly improved coupling efficiencies ranging from 23
to 48% for all investigated substrates (Table 1). The coupling
efficiencies in the reactions with toluene and anisole very high
for a P450 monooxygenase (48%).[13] Coupling efficiencies of
M2 for bromo- and chlorobenzene were improved by six- and
fivefold, making P450 BM3M2 a very efficient catalyst for
converting substituted benzenes. A semi-preparative-scale
conversion of anisole and toluene with 10À4 mol% P450 BM3
M2 yielded the respective phenolic products at levels of
0.67 gLÀ1 (6195 TTN) 0.31 gLÀ1 (2870 TTN).
Received: May 9, 2013
Published online: July 1, 2013
Table 1 reveals that product formation rates and coupling
efficiencies differ significantly in contrast to the regioselec-
Keywords: hydroxylation · monooxygenases · P450 · phenol ·
regioselectivity
.
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