Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Article abstract of DOI:10.1002/anie.201703461

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min^?1 P450BM3^?1 and 40 200 P450BM3^?1 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

Full text of DOI:10.1002/anie.201703461

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Direct Hydroxylation of Benzene to Phenol by Cytochrome
P450BM3 Triggered by Amino Acid Derivatives
Authors: Osami Shoji, Sota Yanagisawa, Joshua Kyle Stanfield,
Kazuto Suzuki, Zhiqi Cong, Hiroshi Sugimoto, Yoshitsugu
Shiro, and Yoshihito Watanabe
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703461
Angew. Chem. 10.1002/ange.201703461
Link to VoR: http://dx.doi.org/10.1002/anie.201703461
http://dx.doi.org/10.1002/ange.201703461

10.1002/anie.201703461
Angewandte Chemie International Edition
COMMUNICATION
Direct Hydroxylation of Benzene to Phenol by Cytochrome
P450BM3 Triggered by Amino Acid Derivatives
Osami Shoji,[b] [c]* Sota Yanagisawa,[b] Joshua Kyle Stanfield,[b] Kazuto Suzuki,[b] Zhiqi Cong,[b] Hiroshi
Sugimoto,[c] [d] Yoshitsugu Shiro,[d] and Yoshihito Watanabe [a]*
Abstract: Selective hydroxylation of benzene to phenol, without
formation of side products resulting from overoxidation, was catalyzed
by cytochrome P450BM3 (P450BM3) with the assistance of amino
acid derivatives as decoy molecules. The catalytic turnover rate and
the total turnover number reached 259 min–1P450BM3–1 and 40,200
dummy substrates (decoy molecules).[5] [6] These PFCs mimic the
native fatty acyl substrate and are capable of erroneously
inducing the activation of molecular oxygen and subsequent
generation of compound I,[7] which due to the inert nature of the
C–F bonds (C–F bond energy)[8] of the PFC leads to the
hydroxylation of gaseous alkanes and benzene (Fig. 1b).
Considering that alkyl carboxylic acids with shorter alkyl chains,
such as nonanoic acid (C9), cannot serve as decoy molecules, it
was concluded that fluorination of carboxylic acids is critical for
decoy molecule function.
P450BM3–1 when N-heptyl-
L-proline modified with L-phenyl alanine
(C7- -Pro- -Phe) was used as the decoy molecule. This work shows
L
L
that amino acid derivatives that have a totally different structure from
fatty acids can be used as decoy molecules for aromatic hydroxylation
by wild-type P450BM3. This methodology for nonnative substrate
hydroxylation by wild-type P450BM3 has the potential to expand the
utility of P450BM3 for biotransformations.
Enzymes catalyze a wide variety of reactions under very mild
conditions, and have therefore been regarded as promising green
catalysts for producing pharmaceuticals, fine chemicals, and
biofuels. Developing a catalyst for selective oxyfunctionalizations
is a longstanding challenge and a current topic of interest.[1]
Cytochrome P450s (P450s) are regarded as potential candidates
for the development of biocatalysts because of their high catalytic
activity in the hydroxylation of unactivated C–H bonds.[2]
A
plethora of engineered P450s have thus been constructed by
mutagenesis and chemical modifications.[3] Among the reported
P450s, CYP102A1 (P450BM3) isolated from Bacillus megaterium
has garnered much attention because of its high monooxygenase
activity.[4] In general, P450BM3 displays a high substrate
specificity, exclusively catalyzing the hydroxylation of long-alkyl-
chain fatty acids (Fig. 1a) while remaining inactive for small non-
native substrates such as propane and benzene. However, it was
observed that P450BM3 can be “fooled” into initiating
hydroxylation of non-native substrates in the presence of
perfluorinated carboxylic acids (PFCs), which function as inert
Figure 1. a) General reaction mechanism of fatty acid hydroxylation catalyzed
by P450BM3. b) A plausible reaction mechanism of benzene hydroxylation
catalyzed by P450BM3 with perfluorononanoic acid (PFC9) as a decoy
[a]
[b]
Prof. Dr. Y. Watanabe
molecule. c) The active site structure of P450BM3 with N-perfluorononanoyl-
tryptophan (PFC9- -Trp). The distance between the heme iron atom and the
terminal carbon atom of PFC9- -Trp is shown by a blue dashed line.
L-
Research Center for Materials Science, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81) 52-789-2953
E-mail: p47297a@nucc.cc.nagoya-u.ac.jp
Dr. O. Shoji, S. Yanagisawa, J. K. Stanfield, K. Suzuki, Z. Cong
Department of Chemistry, Graduate School of Science
Nagoya University
L
L
Recently, we succeeded in enhancing the catalytic activity
of P450BM3 for gaseous alkanes by developing a series of
second-generation decoy molecules, namely N-perfluoroacyl
amino acids.[9] For example, propane was efficiently hydroxylated
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81) 52-789-3557
by P450BM3 with the assistance of N-perfluorononanoyl-
leucine (PFC9- -Leu). Crystal structure analysis of the N-
perfluorononanoyl- -tryptophan (PFC9- -Trp)-bound form of
L-
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
Dr. O. Shoji, Dr. H. Sugimoto
Core Research for Evolutional Science and Technology, Japan
Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo,
102-0075, Japan
L
[c]
[d]
L
L
P450BM3 (PDB code: 3WSP) revealed that the alkyl chain
terminus does not fully penetrate into the active site owing to
multiple hydrogen-bonding interactions between the carboxyl and
Prof. Dr. Y. Shiro, Dr. H. Sugimoto
RIKEN SPring-8 Center
Harima Institute
1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan)
carbonyl groups of PFC9-L-Trp and the amino acids (Tyr-51, Gln-
73, and Ala-74) located at the entrance of the active site of
P450BM3 (Fig. 1c). Therefore, we hypothesized that the alkyl
chain terminus of nonanoic acid (C9) modified with tryptophan
Supporting information for this article is available on the WWW
under http://www.angewandte.org.
This article is protected by copyright. All rights reserved.

10.1002/anie.201703461
Angewandte Chemie International Edition
COMMUNICATION
(C9-L-Trp) would be sufficiently distant from the heme iron to
prevent hydroxylation by P450BM3, even in the absence of
fluorination. If the hydroxylation of carboxylic acids were to be
suppressed by modification with amino acids, diverse carboxylic
acids without the requirement for fluorination could be exploited
as building blocks for novel decoy molecules. Herein, we report
that various carboxylic acids modified with amino acids (N-acyl
amino acids) as well as amino acid dipeptides (Fig. 2) can serve
as decoy molecules for P450BM3. Benzene was more efficiently
Table 1. Turnover rate and coupling efficiency of benzene hydroxylation[a]
Decoy molecule
PFC9
Rate [/min/P450][b]
Coupling efficiency[c] [%]
120 ± 5 Ref. [6b]
134 ± 9
157 ± 4
n.d.
19
29
31
n.d.
41
11
8
PFC9-
PFC9-
Nonanoic acid (C9)
C9- -Trp
C9-Gly
C9- -Ala
L-Trp
L-Phe
hydroxylated in the presence of N-heptyl-
L-proline modified with
L
-phenylalanine (C7- -Pro- -Phe, Fig. 2) than with any other
L
L
L
169 ± 10
9 ± 1
decoy molecule. The maximum turnover rate and coupling
efficiency ([phenol]/[NADPH consumption]100) were 259 min–
1P450BM3–1 and 43%, respectively, and the total turnover number
(TON) reached 40,200 ± 1,700 P450–1.
L
12 ± 1
C9-
C9-
L
-Met
90 ± 5
39
39
13
41
38
20
44
45
19
36
40
47
41
43
L-Leu
60 ± 6
C9-
D
-Leu
9 ± 1
C9-
L
-Phe
192 ± 8
104 ± 6
38 ± 3
C9-
D
-Phe
C8-
L
-Phe
C10-
C11-
L
-Phe
201 ± 11
175 ± 7
51 ± 4
L
-Phe
Ph-C6-
R-Ibu-
L-Phe
L
-Phe
-Phe
-Phe
225 ± 9
236 ± 4
64 ± 2
S-Ibu-
L
Z-Gly-
L
Z-
L-Pro-
L-Phe
229 ± 9
259 ± 12
C7-
L-Pro-
L-Phe
[a] Reaction conditions: P450BM3 (0.5 M), decoy molecule (100 M), benzene
(10 mM), NADPH (5 mM). [b] Uncertainty is given as the standard deviation of
at least three measurements. [c] ([Phenol]/[NADPH consumption])100. n.d. not
detected.
function in the case of P450BM3 can be dismissed. To investigate
the effect of the amino acid side chain, a series of nonanoic acid
derivatives modified with amino acids (C9-L-amino acids) were
prepared. The catalytic turnover rate was highly dependent upon
the type of amino acid modification, and C9 derivatives modified
with
tendency to exhibit higher catalytic activities. C9-
the highest turnover rate (192 min–1) of all C9-
examined in this study. It is important to note here that
modification with -amino acids (C9- -Phe and C9- -Leu) was
superior to modification with -amino acids (C9- -Phe and C9-
L
-amino acids possessing larger side chains displayed a
-Phe afforded
-amino acids
L
L
L
L
L
D
D
D-
Leu), indicating that amino acid chirality also affects catalytic
activity. Catalytic activity was also dependent upon the decoy
molecule’s alkyl chain length, with C9-L-Phe and C10-L-Phe
Figure 2. Structure of decoy molecules.
delivering better turnover rates than longer or shorter variants in
this series. The UV-Vis spectral perturbations of P450BM3
recorded upon the addition of N-acyl amino acids were very small
(Fig. S2), indicating a lack of a clear spin transition of the heme
iron, rendering the determination of reliable dissociation constants
(Kd) for N-acyl amino acids by UV-Vis titration experiment
challenging. We tentatively estimated Kd values for binding of N-
acyl amino acids from the very small observed spectral
perturbations obtained (Fig. S2); however, no decisive differences
could be observed, with all Kd values clustered in a similar range
(8–41 M). Although the UV-Vis spectral perturbations upon
addition of N-acyl amino acids were not clear, we presumed that
N-acyl amino acids bind to the active site in a similar manner to
Initially, we examined the possibility of using nonanoic acid
modified with tryptophan (C9-L-Trp) and leucine (C9-L-Leu) as
decoy molecules for benzene hydroxylation. In the presence of
these decoy molecules, benzene was hydroxylated to phenol
without formation of any side products such as catechol and
hydroquinone resulting from overoxidation of phenol, (Table 1 and
Fig. S1), while no product was detected when bare nonanoic acid
(C9) was used as a decoy molecule. The catalytic turnover rate of
phenol formation reached 169 min–1P450–1 in the case of C9-
Trp, which was higher than that for perfluorinated PFC9- -Trp at
L-
L
134 min–1P450–1 (Table 1). These results clearly indicate that the
simple modification of the nonanoic acid carboxylate moiety with
tryptophan is sufficient to convert it into an effective decoy
molecule. Furthermore, these findings also show that the previous
assumption that fluorination is required for decoy molecule
that observed with PFC9-L-Trp (Fig. 1c). This assumption is
supported by reports describing the non-native substrates
propranolol and propylbenzene, for which the spin transition of the
This article is protected by copyright. All rights reserved.

10.1002/anie.201703461
Angewandte Chemie International Edition
COMMUNICATION
heme iron of P450BM3 upon addition of these substrates was
also ill-defined, despite their being oxidized by P450BM3.[10]
Consistent with the small spectral perturbations seen upon
addition of N-acyl amino acids, the consumption of NADPH in the
improve the turnover rate of benzene hydroxylation. Amino acid
dipeptides containing a carboxybenzyl protecting group (Z-
protection group) were also prepared as potential decoy
molecules. Interestingly, ibuprofen modified with phenylalanine (R
presence of C9-
PFC9- -Phe. However, NADPH was consumed rapidly when
benzene was added simultaneously (Fig. 3a). These results
clearly showed that C9- -Phe alone was not sufficient to activate
P450BM3, but that P450BM3 was fully activated in the presence
of both C9- -Phe and benzene. This may be one reason for the
increased coupling efficiency of C9- -Phe (41%) compared with
that of PFC9-
-Phe (29%). Formation of the CO adduct (CO-Fe2+)
using a CO-saturated buffer was also accelerated in the presence
of both C9- -Phe and benzene (Fig. 3b), indicating that heme
L
-Phe alone was significantly slower than with
and S-Ibu-
catalytic activities (Table 1), while Z-Gly-
were subpar. We also examined the hydroxylation of toluene and
anisole by P450BM3, employing S-Ibu- -Phe as the decoy
molecule (Table 2), wherein the ortho-position of monosubstituted
benzenes was selectively hydroxylated. The turnover rates for
hydroxylation of toluene and anisole were 641 and 623 min–
1P450–1 with high coupling efficiencies (62% and 58%). This
coupling efficiency was superior to that of the engineered
P450BM3 variant (R47S/Y51W/I401M, 48% for toluene and 44%
for anisole), albeit the initial turnover rate was inferior to that of
the mutant.[11] The turnover rate for aromatic hydroxylation of
L
-Phe) and Z-
L
-Pro-
L
-Phe showed relatively increased
L
L
-Phe and Ph-C6- -Phe
L
L
L
L
L
L
L
reduction from Fe3+ to Fe2+ was synergistically accelerated by
both decoy molecule and benzene binding to the active site
simultaneously.
monosubstituted benzenes with S-Ibu-L-Phe exceeded that of
PFC9 (220 min–1P450–1 for toluene, 260 min–1P450–1 for
anisole).[6b]
Table 2. Turnover rate and coupling efficiency of hydroxylation of toluene and
anisole using S-Ibu-L
-Phe as the decoy molecule[a]
Rate
[min–1P450–1
Coupling
efficiency[c] [%]
Ortho-selectivity
Substrate
[b]
]
[%]
Toluene
(Ph-CH3)
641 ± 22
62
96
Anisole
(Ph-OCH3)
623 ± 27
58
92
[a] Reaction conditions: P450BM3 (0.5 M), S-Ibu-
L-Phe (100 M), benzene
(10 mM), NADPH (5 mM). [b] Uncertainty is given as the standard deviation of
at least three measurements. [c] ([Phenol]/[NADPH consumption])100.
Figure 4. X-ray crystal structure of P450BM3 with Z-
5XA3). 2Fo-Fc electron-density map of Z- -Pro- -Phe contoured at the 1.0 
level is shown in light-blue cross-hatching. The distance between the heme iron
L-Pro-L-Phe (PDB code:
Figure 3. a) Time course measurement of NADPH consumption by P450BM3
L
L
with 100 M decoy molecules (PFC9 (green), PFC9-L-Phe (blue), and C9-L-Phe
atom and the terminal carbon atom of Z-
line.
L-Pro-L-Phe is shown as a blue dashed
(red)) in the absence (solid line) and presence (dashed line) of 10 mM benzene
monitored by absorption at 340 nm. The concentrations of P450BM3 and
NADPH were 0.25 M and 250 M, respectively. As a control experiment, the
same amount of dimethyl sulfoxide was added to the reaction without decoy
molecule (black line). b) UV-Vis spectra of P450BM3 in a CO-saturated buffer
To confirm the binding of the decoy molecules, we attempted
to crystallize P450BM3 with C9- -Phe, S-Ibu- -Phe, and Z- -Pro-
-Phe and succeeded in determining the crystal structure of the
Z- -Pro- -Phe-bound form of P450BM3 at 2.2 Å resolution (Fig. 4,
PDB Code: 5XA3). In the fatty acid binding channel of P450BM3,
a clear electron density corresponding to Z- -Pro- -Phe could be
observed. While the structure of Z- -Pro- -Phe differs
considerably from that of a basic fatty acid, it can be observed to
bind in a similar fashion to PFC9- -Trp through hydrogen-bonding
L
L
L
L
with 250 M NADPH and 100 M C9-L-Phe before (black line) and after (red
L
L
line) the addition of 10 mM benzene.
L
L
Given that a myriad of nonfluorinated carboxylic acids can be
employed as building blocks for decoy molecules, we prepared
different types of decoy molecules possessing a phenyl group
using carboxylic acids with a length equivalent to C10-L-Phe. We
expected that a potential – stacking interaction between
L
L
L
interactions with three amino acids, Tyr51, Gln73, and Ala74 (Fig.
4). The distance between the terminal carbon atom of the phenyl
benzene and the phenyl ring of the decoy molecules would
This article is protected by copyright. All rights reserved.

10.1002/anie.201703461
Angewandte Chemie International Edition
COMMUNICATION
Keywords: benzene hydroxylation
molecule N-acyl amino acids
• cytochrome P450 • decoy
group of Z-L-Pro-L-Phe and the heme iron was 8.4 Å (Fig. 4).
•
Docking simulations of benzene calculated using AutoDock Vina
(Fig. S3) revealed that there is adequate space for benzene
binding at the distal side of the heme prosthetic group, even after
Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))
binding of Z-
hydroxylation of benzene by P450BM3 in the presence of Z-
-Phe, while Z- -Pro- -Phe remains unhydroxylated (Fig. S4).
It is noteworthy that the proline side chain of Z- -Pro- -Phe
occupied the space around Pro25 (Fig. 4). We presume that the
bulkiness of proline at the center of Z- -Pro- -Phe may play a
crucial role in fixing Z- -Pro- -Phe at the entrance of the fatty acid
binding channel to create adequate space at the distal side of the
heme for benzene binding. In fact, Z-Gly- -Phe and Ph-C6- -Phe
did not efficiently activate P450BM3 for benzene hydroxylation,
while S-Ibu- -Phe, which possesses a bulky phenyl group at the
center of its structure, promoted a catalytic activity that was many
times higher. Simulated structures of S-Ibu- -Phe bound to
L-Pro-L-Phe. These observations are consistent with
L-Pro-
L
L
L
L
L
[1]
[2]
[3]
a) D. Holtmann, M. W. Fraaije, I. W. C. E. Arends, D. J. Opperman, F.
Hollmann, Chem. Commun. 2014, 50, 13180-13200; b) F. Nastri, M.
Chino, O. Maglio, A. Bhagi-Damodaran, Y. Lu, A. Lombardi, Chem. Soc.
Rev. 2016, 45, 5020-5054.
a) P. R. Ortiz de Montellano, Chem. Rev. 2010, 110, 932-948; b) I. G.
Denisov, T. M. Makris, S. G. Sligar, I. Schlichting, Chem. Rev. 2005, 105,
2253-2277; c) B. Meunier, S. P. de Visser, S. Shaik, Chem. Rev. 2004,
104, 3947-3980.
a) C. J. C. Whitehouse, S. G. Bell, L. L. Wong, Chem. Soc. Rev. 2012,
41, 1218-1260; b) S. Kille, F. E. Zilly, J. P. Acevedo, M. T. Reetz, Nat.
Chem. 2011, 3, 738-743; c) R. Singh, J. N. Kolev, P. A. Sutera, R. Fasan,
ACS Catal. 2015, 5, 1685-1691; d) K. D. Zhang, B. M. Shafer, M. D.
Demars, H. A. Stern, R. Fasan, J. Am. Chem. Soc. 2012, 134, 18695-
18704; e) R. Fasan, ACS Catal. 2012, 2, 647-666; f) P. S. Coelho, E. M.
Brustad, A. Kannan, F. H. Arnold, Science 2013, 339, 307-310; g) M.
Erkelenz, C. H. Kuo, C. M. Niemeyer, J. Am. Chem. Soc. 2011, 133,
16111-16118; h) O. Shoji, T. Fujishiro, K. Nishio, Y. Kano, H. Kimoto, S.-
C. Chien, H. Onoda, A. Muramatsu, S. Tanaka, A. Hori, H. Sugimoto, Y.
Shiro, Y. Watanabe, Catal. Sci. Technol. 2016, 6, 5806-5811; i) H. Onoda,
O. Shoji, Y. Watanabe, Dalton Trans. 2015, 44, 15316-15323; j) C. F.
Butler, C. Peet, A. E. Mason, M. W. Voice, D. Leys, A. W. Munro, J. Biol.
Chem. 2013, 288, 25387-25399; k) P. Le-Huu, T. Heidt, B. Claasen, S.
Laschat, V. B. Urlacher, ACS Catal. 2015, 5, 1772-1780.
L
L
L
L
L
L
L
L
P450BM3 showed that the phenyl group including the methyl
group next to the carbonyl moiety of ibuprofen may be located in
proximity to Pro25 (Fig. S5a), similarly to the proline side chain of
Z-
and Ph-C6-
be key for an efficient decoy molecule. Accordingly, we prepared
N-heptyl- -proline modified with -phenylalanine (C7- -Pro- -Phe,
Fig. 2) to further enhance catalytic activity. As expected, the
turnover rate of C7- -Pro-
-Phe reached 259 min–1P450–1 with a
L-Pro-L-Phe. Judging from the comparison between C10-
L-Phe
L-Phe, the terminal alkyl chain of C10- -Phe may also
L
L
L
L
L
L
L
[4]
[5]
[6]
a) S. S. Boddupalli, B. C. Pramanik, C. A. Slaughter, R. W. Estabrook, J.
A. Peterson, Arch. Biochem. Biophys. 1992, 292, 20-28; b) L. O. Narhi,
A. J. Fulco, J. Biol. Chem. 1986, 261, 7160-7169.
coupling efficiency of 43%. To our knowledge, this represents the
highest turnover frequency (TOF) for benzene hydroxylation to
phenol of any biocatalyst reported.[12] Docking simulations further
supported the assumption that P450BM3 can accommodate C7-
a) N. Kawakami, O. Shoji, Y. Watanabe, Angew. Chem. Int. Ed. 2011,
50, 5315-5318; b) F. E. Zilly, J. P. Acevedo, W. Augustyniak, A. Deege,
U. W. Hausig, M. T. Reetz, Angew. Chem. Int. Ed. 2011, 50, 2720-2724.
a) N. Kawakami, O. Shoji, Y. Watanabe, Chem. Sci. 2013, 4, 2344-2348;
b) O. Shoji, T. Kunimatsu, N. Kawakami, Y. Watanabe, Angew. Chem.
Int. Ed. 2013, 52, 6606-6610; c) N. Kawakami, Z. Cong, O. Shoji, Y.
Watanabe, J. Porphyrins Phthalocyanines 2015, 19, 329-334; d) O. Shoji,
Y. Watanabe, Isr. J. Chem. 2015, 55, 32-39.
L
-Pro-
Finally, we estimated the TON for benzene hydroxylation using
C7- -Pro- -Phe as a decoy molecule. The TON for a 12-h reaction
L-Phe in a similar fashion to Z-L-Pro-L-Phe (Fig. S5b).
L
L
using 25 nM P450BM3 reached 40,200 ± 1,700 with a coupling
efficiency of 46%.
[7]
[8]
[9]
J. Rittle, M. T. Green, Science 2010, 330, 933-937.
In conclusion, we have demonstrated that N-acyl amino acids
as well as amino acid dipeptides strongly activate P450BM3 for
benzene hydroxylation to generate phenol without formation of
side products due to overoxidation of phenol, even though we did
not perform any mutagenesis of P450BM3. We believe that the
direct hydroxylation of benzene to phenol by P450BM3 assisted
by the simple addition of amino acid derivatives as presented here
could be an attractive alternative for phenol production. Despite
having examined only a limited number of carboxylic acids and
amino acids for the preparation of decoy molecules, further
screening is expected to enhance the catalytic activity of
P450BM3. We believe that the catalytic turnover rate and coupling
efficiency for benzene hydroxylation can be improved further by
optimizing the decoy molecule structure based upon the crystal
R. E. Banks, J. C. Tatlow, J. Fluorine Chem. 1986, 33, 227-346.
Z. Cong, O. Shoji, C. Kasai, N. Kawakami, H. Sugimoto, Y. Shiro, Y.
Watanabe, ACS Catal. 2015, 5, 150-156.
[10] a) C. J. C. Whitehouse, S. G. Bell, H. G. Tufton, R. J. P. Kenny, L. C. I.
Ogilvie, L.-L. Wong, Chem. Commun. 2008, 966-968; b) V. E. Ferrero,
G. Di Nardo, G. Catucci, S. J. Sadeghi, G. Gilardi, Dalton Trans. 2012,
41, 2018-2025.
[11] A. Dennig, N. Lulsdorf, H. F. Liu, U. Schwaneberg, Angew. Chem. Int.
Ed. 2013, 52, 8459-8462.
[12] a) R. S. Wang, T. Nakajima, H. Tsuruta, T. Honma, Chem. Biol. Interact.
1996, 99, 239-252; b) D. R. Koop, C. L. Laethem, G. G. Schnier, Toxicol.
Appl. Pharmacol. 1989, 98, 278-288; c) E. T. Farinas, M. Alcalde, F.
Arnold, Tetrahedron 2004, 60, 525-528.
[13] S. D. Munday, O. Shoji, Y. Watanabe, L.-L. Wong, S. G. Bell, Chem.
Commun. 2016, 52, 1036-1039.
structure of P450BM3 with Z-L-Pro-L-Phe, and in combination with
mutagenesis to further tailor the active site for this reaction.[13]
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
(S) to Y. W. (24225004) from the Ministry of Education, Culture,
Sports, Science, and Technology (Japan) and JST CREST Grant
Number JPMJCR15P3, Japan. This work was also supported by
JSPS KAKENHI Grant Number JP15H05806 in Precisely
Designed Catalysts with Customized Scaffolding to O. S.
This article is protected by copyright. All rights reserved.

10.1002/anie.201703461
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Amino acid derivatives whose
Osami Shoji*, Sota Yanagisawa,
structures are totally different from
fatty acids efficiently activate
Joshua Kyle Stanfield, Kazuto Suzuki,
Zhiqi Cong, Hiroshi Sugimoto,
Yoshitsugu Shiro, and Yoshihito
Watanabe*
cytochrome P450BM3 for direct
hydroxylation of benzene to phenol.
The catalytic turnover rate and total
turnover number reached 259 min–
Page No. – Page No.
1P450BM3–1 and 40,200 P450BM3–1
.
Direct Hydroxylation of Benzene to
Phenol by Cytochrome P450BM3
Triggered by Amino Acid Derivatives
This article is protected by copyright. All rights reserved.