Highly selective hydroxylation of benzene to phenol by wild-type cytochrome P450BM3 assisted by decoy molecules

Article abstract of DOI:10.1002/anie.201300282

Playing tricks on enzymes: Direct hydroxylation of benzene to phenol was catalyzed by wild-type P450BM3 in the presence of perfluorinated carboxylic acids as decoy molecules. The catalytic turnover rate reached 120 min ^-1 per P450. The selectivity towards phenol production was very high and no overoxidation products were detected. Copyright

Full text of DOI:10.1002/anie.201300282

Angewandte
Chemie
DOI: 10.1002/anie.201300282
Direct Benzene Hydroxylation
Highly Selective Hydroxylation of Benzene to Phenol by Wild-type
Cytochrome P450BM3 Assisted by Decoy Molecules**
Osami Shoji,* Tatsuya Kunimatsu, Norifumi Kawakami, and Yoshihito Watanabe*
Phenol is a key intermediate in industry for the synthesis of
drugs, dyes, and functional polymers. Because phenol is
currently produced by the cumene process,[1] which involves
high energy consumption and significant formation of side
products such as acetone and methylstyrene, direct hydrox-
ylation of benzene has attracted much attention as an
alternative method of production. The direct hydroxylation
of benzene to phenol using a variety of catalysts,[2] electro-
chemical oxidation systems,[3] and photochemical systems[4]
has thus been extensively investigated. In contrast to many
oxidation reactions at high temperature, monooxygenases in
nature, such as cytochrome P450, efficiently catalyze the
oxidation of inert alkanes and aromatic compounds under
mild conditions.[5] Thus, various engineered enzymes have
been constructed by site-directed mutagenesis, random muta-
genesis, and chemical modification.[6] Biocatalysts for exclu-
sive hydroxylation of the benzene ring using natural enzymes,
if they could be developed, would be ideal systems for the
production of phenol. Herein we report an efficient and
selective hydroxylation of benzene to phenol catalyzed by
wild-type cytochrome P450BM3 (P450BM3)[7] with the assis-
tance of decoy molecules.[8]
the presence of perfluorinated carboxylic acids (PFCs) as
inert dummy substrates (decoy molecules), gaseous alkanes
can be hydroxylated by wild-type P450BM3. The decoy
molecules initiate the activation of molecular oxygen in the
same manner as long-alkyl-chain fatty acids and induce the
generation of compound I[12] (Figure 1b, right). Because the
[13]
À
C F bonds of PFCs are not oxidizable,
compound I
exclusively hydroxylates gaseous alkanes. Moreover, short-
alkyl-chain PFCs partially occupy the substrate binding site of
P450BM3 to afford a space for small alkanes, which leads to
efficient hydroxylation of the small alkanes (Figure 1a,
lower). This attractive advantage encouraged us to perform
benzene hydroxylation for the selective production of phenol.
The hydroxylation of benzene by P450BM3 was examined
by employing a series of PFCs (PFC8–PFC12; Table 1). The
catalytic turnover rates of phenol formation were very much
Table 1: Turnover rate, NADPH consumption, and coupling efficiency of
benzene hydroxylation,[a] and the dissociation constant of decoy
molecules.[b]
We and Zilly et al. have recently developed a simple and
unique system for the hydroxylation of gaseous alkanes, such
as propane and butane, catalyzed by wild-type P450BM3.[9]
Wild-type P450BM3 exclusively catalyzes the hydroxylation
of long-alkyl-chain fatty acids and never hydroxylates small
alkanes, because the active site of P450BM3 is optimized for
the hydroxylation of fatty acids (Figure 1a, upper)[10] and the
first step of the catalytic cycle of P450BM3 starts only when
a fatty acid binds to the substrate binding site of P450BM3,
which is accompanied by the removal of the water molecule
coordinated to the heme iron (Figure 1b, left).[11] However, in
Decoy[c]
Rate[d]
NADPH
Coupling
Kd [mm][f]
consumption[d]
efficiency [%][e]
PFC8
PFC9
PFC10
PFC11
PFC12
none
38Æ6
120Æ9
120Æ5
83Æ6
71Æ5
n.d.
230Æ41
500Æ13
600Æ39
560Æ17
590Æ26
–
17
24
19
15
12
–
1900
980
290
91
30
–
[a] Reaction conditions: P450BM3 (0.5 mm), decoy molecule (100 mm),
benzene (10 mm), NADPH (5 mm). [b] from Ref. [9a]. [c] Decoy com-
pounds are all linear perfluorinated carboxylic acids; the number shown
indicates the chain length. [d] Rates and NADPH consumption are given
in units of [minÀ1 per P450]. Uncertainty is given as the standard
deviation from at least three measurements. [e] ([Phenol]/[NADPH
consumption])ꢀ100. [f] Kd =dissociation constant. n.d.=not detected.
[*] Dr. N. Kawakami, Prof. Dr. Y. Watanabe
Research Center for Materials Science, Nagoya University, Furo-cho
Chikusa-ku, Nagoya, 464-8602 (Japan)
E-mail: p47297a@nucc.cc.nagoya-u.ac.jp
Homepage: http://bioinorg.chem.nagoya-u.ac.jp/
Dr. O. Shoji, T. Kunimatsu
Department of Chemistry, Graduate School of Science, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
dependent on the alkyl chain length of the PFCs. PFC9 and
PFC10 afforded the highest turnover rate, 120 minÀ1 per
P450.[14] Without PFCs, this reaction did not proceed at all.
The coupling efficiency (defined as ([phenol]/[NADPH con-
sumption]) ꢀ 100) of PFC9 (24%) was the highest among the
PFCs examined, indicating that the active site provided by
PFC9 is suitable for the accommodation of benzene.
Although hydroxylation of benzene is generally accompanied
by further oxidation of phenol,[15] we observed exclusive
formation of phenol without any overoxidation products. The
[**] This work was supported by Grants-in-Aid for Scientific Research (S)
to Y.W. (24225004) and a Grant-in-Aid for Young Scientists (A) to
O.S. (21685018) from the Ministry of Education, Culture, Sports,
Science, and Technology (Japan). A plasmid encoding P450BM3
was kindly supplied by Prof. Stephen G. Sligar, University of Illinois
(USA).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300282.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
Figure 1. a) The active site of P450BM3 containing palmitoleic acid (upper; PDB code: 1FAG) and the proposed active-site structure of P450BM3
containing perfluorononanoic acid (PFC9) and benzene (lower). b) General reaction mechanism of P450BM3 (left) and the reaction mechanism of
propane and benzene hydroxylation catalyzed by P450BM3 with PFC9 as a decoy molecule (right).
selectivity towards phenol production was more than 99%,
according to high-performance liquid chromatography
(HPLC) analysis (see the Supporting Information). Phenol
is expected to escape rapidly from the active site of P450BM3
because the heme cavity consists of hydrophobic amino acid
residues (see the Supporting Information) and the hydro-
phobic nature of PFC. In fact, phenol was less reactive than
benzene under the same conditions.[16] Furthermore, only
small amounts of hydroquinone and catechol were detected
when phenol (10 mm) was added as a substrate (see the
Supporting Information). The turnover rate and coupling
efficiency of the benzene hydroxylation are higher than those
of a P450BM3 mutant (28 minÀ1 per P450, 14%) developed
Scheme 1. Proposed reaction mechanism for benzene hydroxylation by
by random mutagenesis.[17] Although there are a few reports
on the catalytic turnover rate for benzene hydroxylation
catalyzed by isolated P450s,[18] to the best of our knowledge,
this is the first example demonstrating the exclusive forma-
tion of phenol by the hydroxylation of benzene with P450.
The [D6]benzene hydroxylation had a higher turnover rate of
135 minÀ1 per P450 (NADPH consumption of 435 minÀ1 per
P450 and coupling efficiency of 31%; see the Supporting
Information), which indicates that the rate-determining step
P450BM3.[19e]
Because the ortho selectivity in toluene hydroxylation was
not much affected by the length of the PFCs, we assume that
the regioselectivity is mainly controlled by amino acid
residues at the active site of P450BM3. Phe87, which is
located on the distal side of the heme (see the Supporting
Information), could interact with toluene to induce the ortho
position selective hydroxylation. In fact, the ortho selectivity
was reduced in the case of the F87A mutant of P450BM3.[21]
The ortho-selective hydroxylation was also observed in the
hydroxylation of anisole and chlorobenzene (Table 3; see also
the Supporting Information). We did not observe any color
change in the reaction of anisole hydroxylation even though
o-hydroxyanisole (guaiacol) formed. Guaiacol is a substrate
commonly used to examine the oxidation activity of heme
enzymes such as peroxidase by affording its dimer along with
a color change.[22] Moreover, even though the ortho deme-
thylation of anisole is a common reaction for human and rat
hepatic P450s,[23] the ortho demethylation of anisole to give
À
does not include C H bond cleavage, but is rather well
explained by assuming epoxide formation followed by
rearrangement reactions, including a hydride shift, known as
an NIH shift (Scheme 1).[19] The observed deuterium kinetic
isotope effect (< 1) is attributed to the change in carbon
atoms from sp2 to sp3 for the formation of epoxide, which
generally affords an inverse isotope effect.[20]
Toluene was also hydroxylated, and PFC9 gave the
highest turnover rate (220 minÀ1 per P450) for the selective
ortho hydroxylation (Table 2). The coupling efficiency
reached 56%.
2
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Table 2: Turnover rate, NADPH consumption, and coupling efficiency of toluene hydroxylation.[a]
repeated mutagenesis. We believe
that the catalytic turnover rate and
coupling efficiency for benzene hy-
droxylation would be further
improved by optimizing the struc-
ture of decoy molecules to increase
their binding constants toward that
of P450BM3 (Table 1).[9a] More-
over, it would be possible to control
the regioselectivity of the hydroxyl-
ation of benzene derivatives by
changing the structure of decoy
molecules.
Decoy[b]
Rate[c]
p-cresol
NADPH
Coupling
consumption[c]
efficiency [%][d]
o-cresol
m-cresol
benzyl alcohol
PFC8
PFC9
PFC10
PFC11
PFC12
120Æ2
220Æ7
210Æ23
150Æ10
140Æ15
n.d.
n.d.
n.d.
n.d.
n.d.
3.2Æ0.1
5.3Æ0.2
5.0Æ0.7
3.5Æ0.3
3.3Æ0.4
7.7Æ0.5
16Æ0.4
18Æ2
260Æ12
430Æ34
520Æ41
410Æ30
570Æ7
51
56
45
39
27
10Æ1
7.2Æ0.9
[a] Reaction conditions: P450BM3 (0.5 mm), decoy molecule (100 mm), toluene (10 mm), NADPH
(5 mm). [b] Decoy compounds are all linear perfluorinated carboxylic acids; the number shown indicates
the chain length. [c] Rates and NADPH consumption are given in units of [minÀ1 per P450]. Uncertainty
is given as the standard deviation from at least three measurements. [d] ([Products]/[NADPH
consumption])ꢀ100. n.d.=not detected.
Received: January 12, 2013
Revised: March 7, 2013
Published online: && &&, &&&&
Keywords: carboxylic acid ·
.
cytochromes · decoy molecule ·
hydroxylation
Table 3: Turnover rates for the hydroxylation of benzene derivatives.[a]
[1] R. J. Schmidt, Appl. Catal. A 2005, 280, 89 – 103.
[2] a) S.-i. Niwa, M. Eswaramoorthy, J. Nair, A. Raj, N. Itoh, H.
Shoji, T. Namba, F. Mizukami, Science 2002, 295, 105 – 107; b) R.
Bal, M. Tada, T. Sasaki, Y. Iwasawa, Angew. Chem. 2006, 118,
462 – 466; Angew. Chem. Int. Ed. 2006, 45, 448 – 452; c) G. I.
Panov, CATTECH 2000, 4, 18 – 31; d) J. Jia, K. S. Pillai, W. M. H.
Sachtler, J. Catal. 2004, 221, 119 – 126; e) S. Song, H. Yang, R.
Rao, H. Liu, A. Zhang, Appl. Catal. A 2010, 375, 265 – 271; f) T.
Dong, J. Li, F. Huang, L. Wang, J. Tu, Y. Torimoto, M. Sadakata,
Q. Li, Chem. Commun. 2005, 2724 – 2726; g) M. Tani, T.
Sakamoto, S. Mita, S. Sakaguchi, Y. Ishii, Angew. Chem. 2005,
117, 2642 – 2644; Angew. Chem. Int. Ed. 2005, 44, 2586 – 2588.
[3] a) B. Lee, H. Naito, T. Hibino, Angew. Chem. 2012, 124, 455 –
459; Angew. Chem. Int. Ed. 2012, 51, 440 – 444; b) I. Yamanaka,
T. Akimoto, K. Otsuka, Electrochim. Acta 1994, 39, 2545 – 2549;
c) K. Otsuka, I. Yamanaka, K. Hosokawa, Nature 1990, 345,
697 – 698.
Substrate
R=
Rate[b]
meta
ortho
para
OCH3
Cl
NO2
260Æ4
n.d.
n.d.
n.d.
n.d.
38Æ0.3
14Æ0.6
n.d.
120Æ4
0.9Æ0.05
2.9Æ0.1
COCH3
n.d.
[a] Reaction conditions: P450BM3 (0.5 mm), PFc9 (100 mm), substrate
(10 mm), NADPH (5 mm). [b] Rates are given in units of [minÀ1 per
P450]. Uncertainty is given as the standard deviation from at least three
measurements. n.d.=not detected.
phenol and formaldehyde was not observed. Furthermore, the
hydroxylation of the electron-poor benzene derivatives nitro-
benzene and acetophenone gave the o-hydroxylated product,
despite their meta-preference in many reactions. These results
clearly show that the ortho position of monosubstituted
benzenes was selectively hydroxylated irrespective of sub-
stituents. Although co-crystal structure analysis of P450BM3
with PFC9 would be required to clarify the active-site
structure of a P450BM3–PFC9 complex and determine the
possible locations and orientations of benzene and its
derivatives in the heme pocket, we have not yet succeeded
in the co-crystallization.
In conclusion, we have demonstrated selective hydroxyl-
ation of benzene to phenol catalyzed by wild-type P450BM3
through the simple addition of PFCs as decoy molecules.
Although we did not replace any amino acid residues, the
catalytic turnover rate and the coupling efficiency are higher
than those of the engineered P450BM3 mutants prepared by
directed evolution.[17] The benzene hydroxylation reported
here is an example of how a P450BM3–decoy molecule
system can be used to develop biocatalysts as an alternative to
[4] a) K. Ohkubo, T. Kobayashi, S. Fukuzumi, Angew. Chem. 2011,
123, 8811 – 8814; Angew. Chem. Int. Ed. 2011, 50, 8652 – 8655;
b) Y. Shiraishi, N. Saito, T. Hirai, J. Am. Chem. Soc. 2005, 127,
12820 – 12822; c) K.-i. Shimizu, H. Akahane, T. Kodama, Y.
Kitayama, Appl. Catal. A 2004, 269, 75 – 80.
[5] a) P. R. Ortiz de Montellano, 3rd ed., Cytochrome P450:
Structure, Mechanism, and Biochemistry, Plenum, New York,
2005; b) M. Sono, M. P. Roach, E. D. Coulter, J. H. Dawson,
Chem. Rev. 1996, 96, 2841 – 2888; c) I. G. Denisov, T. M. Makris,
S. G. Sligar, I. Schlichting, Chem. Rev. 2005, 105, 2253 – 2278.
[6] a) S. Kille, F. E. Zilly, J. P. Acevedo, M. T. Reetz, Nat. Chem.
2011, 3, 738 – 743; b) R. Agudo, G. D. Roiban, M. T. Reetz,
ChemBioChem 2012, 13, 1465 – 1473; c) R. Fasan, M. M. Chen,
N. C. Crook, F. H. Arnold, Angew. Chem. 2007, 119, 8566 – 8570;
Angew. Chem. Int. Ed. 2007, 46, 8414 – 8418; d) P. Meinhold,
M. W. Peters, M. M. Y. Chen, K. Takahashi, F. H. Arnold,
ChemBioChem 2005, 6, 1765 – 1768; e) F. Xu, S. G. Bell, J.
Lednik, A. Insley, Z. H. Rao, L. L. Wong, Angew. Chem. 2005,
117, 4097 – 4100; Angew. Chem. Int. Ed. 2005, 44, 4029 – 4032;
f) C. J. C. Whitehouse, S. G. Bell, L. L. Wong, Chem. Soc. Rev.
2012, 41, 1218 – 1260; g) A. Dennig, J. Marienhagen, A. J. Ruff,
L. Guddat, U. Schwaneberg, ChemCatChem 2012, 4, 771 – 773;
h) R. Fasan, Y. T. Meharenna, C. D. Snow, T. L. Poulos, F. H.
Arnold, J. Mol. Biol. 2008, 383, 1069 – 1080; i) C. J. C. White-
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
house, S. G. Bell, W. Yang, J. A. Yorke, C. F. Blanford, A. J. F.
Strong, E. J. Morse, M. Bartlam, Z. H. Rao, L. L. Wong,
ChemBioChem 2009, 10, 1654 – 1656; j) E. Weber, A. Seifert,
M. Antonovici, C. Geinitz, J. Pleiss, V. B. Urlacher, Chem.
Commun. 2011, 47, 944 – 946; k) A. Seifert, S. Vomund, K.
Grohmann, S. Kriening, V. B. Urlacher, S. Laschat, J. Pleiss,
ChemBioChem 2009, 10, 853 – 861; l) M. Erkelenz, C. H. Kuo,
C. M. Niemeyer, J. Am. Chem. Soc. 2011, 133, 16111 – 16118;
m) T. Fujishiro, O. Shoji, N. Kawakami, T. Watanabe, H.
Sugimoto, Y. Shiro, Y. Watanabe, Chem. Asian J. 2012, 7,
2286 – 2293; n) O. Shoji, C. Wiese, T. Fujishiro, C. Shirataki, B.
Wꢁnsch, Y. Watanabe, J. Biol. Inorg. Chem. 2010, 15, 1109 –
1115.
phosphate (NADPH; 5 mm) was consumed after 30 min under
the conditions tested.
[15] P. M. Zhai, L. Q. Wang, C. H. Liu, S. C. Zhang, Chem. Eng. J.
2005, 111, 1 – 4.
[16] The phenol hydroxylation was investigated using phenol (5 mm)
as a substrate. The turnover rates were 70 minÀ1 per P450 for
hydroquinone formation and 15 minÀ1 per P450 for catechol
formation (see the Supporting Information).
[17] E. T. Farinas, M. Alcalde, F. Arnold, Tetrahedron 2004, 60, 525 –
528.
[18] 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.
[19] a) G. Guroff, J. W. Daly, D. M. Jerina, J. Renson, B. Witkop, S.
Udenfrie, Science 1967, 157, 1524 – 1530; b) J. Koerts, A. E. M. F.
Soffers, J. Vervoort, A. De Jager, I. M. C. M. Rietjens, Chem.
Res. Toxicol. 1998, 11, 503 – 512; c) D. M. Jerina, J. W. Daly,
Science 1974, 185, 573 – 582; d) K. Korzekwa, W. Trager, M.
Gouterman, D. Spangler, G. H. Loew, J. Am. Chem. Soc. 1985,
107, 4273 – 4279; e) S. P. de Visser, S. Shaik, J. Am. Chem. Soc.
2003, 125, 7413 – 7424.
[7] a) L. O. Narhi, A. J. Fulco, J. Biol. Chem. 1986, 261, 7160 – 7169;
b) S. S. Boddupalli, B. C. Pramanik, C. A. Slaughter, R. W.
Estabrook, J. A. Peterson, Arch. Biochem. Biophys. 1992, 292,
20 – 28.
[8] Decoy molecules are a series of inert dummy substrates having
structural similarity to a natural substrate, in this case, fatty acids.
[9] a) N. Kawakami, O. Shoji, Y. Watanabe, Angew. Chem. 2011,
123, 5427 – 5430; 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. 2011, 123, 2772 – 2776;
Angew. Chem. Int. Ed. 2011, 50, 2720 – 2724.
[10] a) K. G. Ravichandran, S. S. Boddupalli, C. A. Hasemann, J. A.
Peterson, J. Deisenhofer, Science 1993, 261, 731 – 736; b) H. Y.
Li, T. L. Poulos, Nat. Struct. Biol. 1997, 4, 140 – 146.
[11] D. C. Haines, D. R. Tomchick, M. Machius, J. A. Peterson,
Biochemistry 2001, 40, 13456 – 13465.
[20] R. P. Hanzlik, K. H. J. Ling, J. Am. Chem. Soc. 1993, 115, 9363 –
9370.
[21] 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.
[22] a) G. D. DePillis, B. P. Sishta, A. G. Mauk, P. R. Ortiz de Mon-
tellano, J. Biol. Chem. 1991, 266, 19334 – 19341; b) T. Matsui, S.
Ozaki, Y. Watanabe, J. Am. Chem. Soc. 1999, 121, 9952 – 9957;
c) O. Shoji, T. Fujishiro, H. Nakajima, M. Kim, S. Nagano, Y.
Shiro, Y. Watanabe, Angew. Chem. 2007, 119, 3730 – 3733;
Angew. Chem. Int. Ed. 2007, 46, 3656 – 3659.
[23] a) A. P. Li, D. L. Kaminski, A. Rasmussen, Toxicology 1995, 104,
1 – 8; b) R. J. Weaver, S. Thompson, G. Smith, M. Dickins, C. R.
Elcombe, R. T. Mayer, M. D. Burke, Biochem. Pharmacol. 1994,
47, 763 – 773.
[12] J. Rittle, M. T. Green, Science 2010, 330, 933 – 937.
[13] R. E. Banks, J. C. Tatlow, J. Fluorine Chem. 1986, 33, 227 – 346.
[14] In the case of PFC9, the conversion of benzene into phenol after
40 min of reaction was estimated to be 8.4%, based on the
amount of benzene used. The turnover number of the reaction
was 1690. Most of the nicotinamide adenine dinucleotide
4
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Direct Benzene Hydroxylation
Playing tricks on enzymes: Direct hy-
droxylation of benzene to phenol was
catalyzed by wild-type P450BM3 in the
presence of perfluorinated carboxylic
acids as decoy molecules. The catalytic
turnover rate reached 120 minÀ1 per
P450. The selectivity towards phenol
production was very high and no over-
oxidation products were detected.
O. Shoji,* T. Kunimatsu, N. Kawakami,
Y. Watanabe*
&&&& — &&&&
Highly Selective Hydroxylation of
Benzene to Phenol by Wild-type
Cytochrome P450BM3 Assisted by Decoy
Molecules
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!