Enhancedcis- And enantioselective cyclopropanation of styrene catalysed by cytochrome P450BM3 using decoy molecules

Article abstract of DOI:10.1039/d0cc04883f

We report the enhancedcis- and enantioselective cyclopropanation of styrene catalysed by cytochrome P450BM3 in the presence of dummy substrates,i.e.decoy molecules. With the aid of the decoy molecule R-Ibu-Phe, diastereoselectivity for thecisdiastereomers

Full text of DOI:10.1039/d0cc04883f

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Enhanced cis- and enantioselective cyclopropanation of styrene
catalysed by cytochrome P450BM3 using decoy molecules
a
a
a
a
a,b
Kazuto Suzuki, Yuma Shisaka, Joshua Kyle Stanfield, Yoshihito Watanabe, and Osami Shoji
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We report the enhanced cis- and enantioselective cyclopropanation
of styrene catalysed by cytochrome P450BM3 in the presence of
dummy substrates, i.e. decoy molecules. With the aid of the decoy
molecule R-Ibu-Phe, diastereoselectivity for the cis diastereomers
reached 91%, and the enantiomeric ratio for the (1S,2R) isomer
reached 94%. Molecular dynamics simulations underpin the
experimental data, revealing the mechanism of how
enantioselectivity is controlled by the addition of decoy molecules.
Cyclopropane, the smallest of cycloalkanes, possesses high ring
strain, yet is a chemical structure frequently observed in
biologically significant natural products.1 Compounds
containing the cyclopropyl moiety are synthetically valuable
targets in organic chemistry, as such molecules can be
physiologically active and leveraged as pharmaceuticals, e.g.
arylcyclopropylamines such as the platelet aggregation inhibitor
2
ticagrelor. Owing to their pharmaceutical value, synthetic
chemists have explored numerous avenues to yield
cyclopropanes, one of which is the transition metal-catalysed
Fig. 1 (A) Styrene cyclopropanation mediated by a haem-carbene intermediate derived
from EDA as a carbene source. (B) The active site structure of P450BM3 complexed with
a decoy molecule (S-Ibu-Phe) (PDB ID: 6JO1). (C) The chemical structures of the decoy
addition of a carbene derived from a diazo compound to an molecules employed in this study.
3
alkene. Although significant progress has been made in the
development of catalysts for asymmetric cyclopropanation,
diastereo- and enantioselectivities.6 In addition, artificial
enzymes incorporating a metallocomplex, which can impart the
enzyme with near unlimited reactivity, have also been
developed to realise highly stereoselective cyclopropanations.7
As a unique and different kind of approach for enzyme
engineering, our research group has developed a system,
wherein native substrate mimics (decoy molecules) are
harnessed to activate unadulterated wild-type cytochrome
elaborate metal catalysts bearing chiral ligands have had to be
crafted to achieve sufficiently high stereoselectivities.4
Biocatalysts are expected to offer an alternative to the currently
available challenging and burdensome synthetic methods;
however, carbene transfers are abiological reactions and have
not been found to occur in nature. Remarkably, seminal work
by Arnold and co-workers has established that engineered
haemoproteins can be engineered as catalysts for the
stereoselective cyclopropanation of olefin compounds with
8
P450s (CYPs or P450s) for non-native substrate oxidations. Our
latest reports revealed that by using N-acyl amino acids as
decoy molecules, P450BM3, a fatty acid hydroxylase isolated
from Bacillus megaterium, can be compelled into hydroxylating
5
ethyl diazoacetate (EDA) as a carbene source (Fig. 1A). The
Fasan group has substantiated that variants of myoglobin can
be exploited in cyclopropanation reactions, yielding excellent
9
10
small organic compounds, such as benzene and propane,
which does not occur in the absence of decoy molecules.
Furthermore, we have demonstrated that decoy molecules not
only trigger hydroxylation but can also control stereoselectivity;
for example, depending on the structure of the decoy molecule
employed, during the hydroxylation of indane by wild-type
P450BM3 the apparent enantioselectivity could be inverted,
with the enantiomeric excess ranging from 53% (R) with 5CHVA-
a. Department of Chemistry, School of Science, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-0802, Japan.
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
Core Research for Evolutional Science and Technology (CREST), Japan Science and
b.
Technology Agency, 5 Sanban-cho, Chiyoda-ku, Tokyo, 102-0075, Japan
†
Electronic Supplementary Information (ESI) available: See DOI: 10.1039/
x0xx00000x
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Journal Name
selection of decoy molecules employed in this study were
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DOI: 10.1039/D0CC04883F
chosen for their previously established potential in controlling
1
1
the enantioselectivity of benzylic hydroxylation and their
structures are depicted in Fig. 1C. The results of the
cyclopropanation reactions for the T268A mutant are shown in
Table 1. In the absence of a decoy molecule, the T268A mutant
catalysed the cyclopropanation of styrene with 99% trans
selectivity and an enantiomeric ratio (er) of 98% for the (1S,2S)
isomer, whilst the addition of S-Ibu-Phe increased selectivity for
the thermodynamically less favourable cis diastereomer to 27%,
with the er for the (1S,2R) isomer reaching 83% (Fig. 2A and B).
This experimental fact indicates that the decoy molecule
remains bound to the P450BM3 active site during catalysis,
even for abiological reactions, whereby the active site is
reshaped and stereoselectivity consequently changed. However,
the turnover number (TON) was significantly reduced from 597
Fig. 2 Chromatograms of the products of styrene cyclopropanation catalysed by
P450BM3 mutants in the absence/presence of decoy molecules
Phe to 56% (S) with Z-Pro-Phe.11 Inspired by the recent to 21 by addition of the decoy molecule, suggesting that the
successes of other groups in the development of biocatalysts for active site is too tight to comfortably accommodate all three
carbene transfer reactions, we envisioned that the application molecules, i.e. styrene, the iron haem carbene intermediate,
of decoy molecules could be expanded to encompass such and the decoy molecule, simultaneously. From these
abiological cyclopropanations as well as to dictate the observations we can infer that in the case of S-Ibu-Phe being
stereoselectivity thereof. Herein, we report the stereoselective bound to P450BM3, binding of styrene is likely destabilised,
cyclopropanation of styrene catalysed by mutant P450BM3 in thus curbing efficient cyclopropanation. The fact that 5CHVA-
the presence of decoy molecules with EDA as the carbene Phe and Z-Pro-Phe simply decreased the TON whilst not
source.
exerting any appreciable effect upon stereoselectivity implies
Initially, we prepared the T268A mutant of the P450BM3 that these decoy molecules act as weak competitive inhibitors
haem domain and examined the stereoselectivity of the target and, unlike S-Ibu-Phe, do not remain in the active site during
reaction by pairing the T268A mutant with a selection of decoy cyclopropanation.
molecules. The T268A mutant was selected, because threonine
Given the results of the single mutant, we concluded that it
in this position has been reported to be deleterious to the may be necessary to introduce a further mutation to augment
catalysis of carbene transfers in P450BM3 (Fig. 1B).12 The the active site space, allowing P450BM3 to stably incorporate
decoy molecule, iron haem carbene
Table 1 Cyclopropanation of styrene catalysed by P450BM3 mutants in the presence of decoy
intermediate, and styrene, simultaneously.
molecules.a
We decided to mutate Phe87 into alanine,
as this bulky residue is one of the closest to
the haem cofactor and the likeliest culprit
responsible for obstructing styrene
accommodation in the presence of the
decoy molecule. Cyclopropanation of
styrene by the F87A/T268A double mutant
in the presence of R-Ibu-Phe led to
enhanced diastereo- and enantioselectivity
without a significant loss in activity, with
Mutant
T268A
Decoy
None
cis:trans
(1S,2R):(1R,2S)
(1R,2R):(1S,2S)
TON
1:99
39:61
43:57
40:60
76:24
83:17
75:25
86:14
82:18
94:6
2:98
2:98
2:98
3:97
4:96
7:93
7:93
6:94
15:85
11:89
597 ± 16
188 ± 27
231 ± 16
30 ± 4
5
CHVA-Phe
2:98
9
1% cis selectivity and 94% er for the
Z-Pro-Phe
R-Ibu-Phe
S-Ibu-Phe
None
2:98
(
1S,2R) isomer, compared to 81% cis
selectivity and 75% er in the absence of a
decoy molecule (Fig. 2C, D, and Table 1). In
the presence of the decoy molecules tested,
both diastereo- and enantioselectivity
were changed and somewhat improved,
whilst the drops in TON of the double
mutant caused by the addition of decoy
molecules were less than that of the T268A
single mutant. For instance, whereas the
addition of R-Ibu-Phe reduced the TON of
the T268A mutant to about 5% TON, the
F87A/T268A mutant retained about 80%
10:90
27:73
81:19
91:9
21 ± 3
F87A/T268A
350 ± 13
297 ± 14
350 ± 16
283 ± 10
254 ± 11
5
CHVA-Phe
Z-Pro-Phe
R-Ibu-Phe
S-Ibu-Phe
87:13
91:9
90:10
90:10
a
Reaction conditions: 10 mM styrene, 10 mM EDA, 10 mM Na
00 mM HEPES buffer (pH 8.0) at 25°C for 14 hours.
2 2 4
S O , 200 μM decoy molecule, and 5 μM P450BM3 in
1
2
| J. Name., 2012, 00, 1-3
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To
understand
how
the
stereoselectivity
of
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DOI: 10.1039/D0CC04883F
cyclopropanation catalysed by the F87A/T268A mutant was
improved by the addition of R-Ibu-Phe, we conducted molecular
dynamics (MD) simulations of the P450BM3 mutants, wherein
the haem carbene intermediate was modelled into the
structure manually. We monitored the C―C―Fe―N dihedral
angle during 200 ns simulations, and the results revealed with
great clarity that the dihedral angles were completely different
between the T268A and F87A/T268A mutants, reflecting the
inverted cis/trans preference for cyclopropanation between the
two mutants (Fig. 3A, B, and C). These observations confirmed
that, in the T268A mutant, the Phe87 side chain occludes the
active site space, forcing the ester group of the iron-carbene
intermediate to point away from Phe87 (Fig. 3C (a)). The MD
simulation of the F87A/T268A double mutant with R-Ibu-Phe
yielded similar behaviour of the dihedral angle to the simulation
without a decoy molecule, indicating that the orientation of the
ester group in the iron-carbene intermediate is unperturbed by
the presence of a decoy molecule. A successive docking
Fig. 4 (A) The dihedral angle in the haem-carbene intermediate monitored during the
MD simulations. (B) The plots of the dihedral angle of the mutants during the 200 ns MD simulation of styrene into the “averaged” structure extracted
simulations. (C) The haem-carbene intermediate structures of the average structures from the simulation of the F87A/T268A mutant bound to R-Ibu-
extracted from the 200 ns MD simulations. The hydrogen atoms are omitted for clarity.
Phe resulted in styrene adopting
a
pro-(1S,2R)-like
conformation (Fig. 4A). The benzene ring of styrene is
comfortably accommodated in the space created by the F87A
mutation, leaning away from the isobutyl group of R-Ibu-Phe to
avoid steric repulsion. This docking pose is in harmony with the
experimental evidence that formation of the (1S,2R) isomer was
enhanced during cyclopropanation of styrene by the
F87A/T268A mutant in the presence of R-Ibu-Phe (Table 1).
Moreover, in the case of the T268A mutant, the
computationally predicted product configuration of (1S,2S) is
identical to the experimentally observed selectivity. Taken
together, we can conclude that the decoy molecule R-Ibu-Phe
does not influenced the orientation of the carbenoid species but
only that of styrene in the active site, thereby determining how
styrene approaches the carbene species and ultimately
determining the stereoselectivity of the end products.
TON even in the presence of the decoy molecule. It is worth
noting here that regardless of the presence or absence of R-Ibu-
Phe, the TON for the (1S,2R) isomer was almost the same (242
and 213, respectively, calculated from the TONs and the
product distribution), revealing that the decoy molecule simply
suppresses the production of the other stereoisomers and does
not exert a negative effect upon the reaction route to the
(
1S,2R) isomer. The 91% selectivity for the cis diastereomer,
which remain tough and challenging to access by conventional
4
chemical catalysts, is on par with that of BM3-CIS (a P450BM3
mutant containing 13 mutations: V78A, F87V, P142S, T175I,
A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and
E442K) obtained by directed evolution,5 emphasising the
simplicity of our approach.
Fig. 3 The results of the docking simulations of styrene into the average structure in the MD simulation of the (A) F87A/T268A double mutant with R-Ibu-Phe and (B) T268A single
mutant. (Left) The best docking pose of styrene obtained by the docking simulations. The cavity incorporating styrene is displayed as a surface model. (Right) The predicted product
configurations judging from the orientation of styrene and the haem-carbene intermediate. The hydrogen atoms on the carbene carbon are modelled for clarity.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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6
7
(a) M. Bordeaux, V. Tyagi and R. Fasan, Angew. Chem.VIienwt.AErtdic.l,e2O0n1lin5e,
4, 1744–1748. (b) A. Tinoco, Y. Wei, J.-P. Bacik, D. M. Carminati,
In conclusion, we have disclosed that decoy molecules can
be utilised as auxiliaries to modulate the secondary
5
DOI: 10.1039/D0CC04883F
E. J. Moore, N. Ando, Y. Zhang and R. Fasan, ACS Catal., 2019, 9,
514–1524. (c) D. A. Vargas, R. L. Khade, Y. Zhang and R. Fasan,
13
coordination sphere of the P450BM3 active centre to tune
stereoselectivity for carbene-mediated cyclopropanations, a
reaction type not found to exist in nature. This adds a further
milestone to the chemical transformations compatible with our
decoy molecule system, greatly expanding their versatility.
Moreover, merely two mutations and a decoy molecule were
required to deliver highly cis- and enantioselective
cyclopropanation, something that has only been traditionally
achieved by laborious directed evolution or extensive screening
from nature. The work highlighted herein has successfully
appended decoy molecules to the repertoire of enzyme
engineering techniques available to modulate the
stereoselectivity of cyclopropanation. Molecular dynamics
simulations of the F87A/T268A mutant with R-Ibu-Phe hinted at
the mechanism governing enhanced selectivity, and we believe
this may contribute to the future design of P450BM3-based
cyclopropanation catalysts in conjunction with decoy molecules.
Although still in its infancy, the present study promises the
exploitation of the decoy molecule system to achieve a more
selective catalysis, even for a panel of abiological reactions.
Various types of carbene and nitrene transfer reactions, besides
cyclopropanations, have been showcased by other research
1
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Chandgude and R. Fasan, ACS Catal., 2020, 10, 2308–2313.
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and T. R. Ward, Catal. Sci. Technol., 2018, 8, 2294–2298.
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and Y. Watanabe, Angew. Chem. Int. Ed., 2007, 46, 3656–3659.
8
9
(
b) N. Kawakami, O. Shoji and Y. Watanabe, Angew. Chem. Int.
Ed., 2011, 50, 5315–5318. (c) O. Shoji, T. Kunimatsu, N. Kawakami
and Y. Watanabe, Angew. Chem. Int. Ed., 2013, 52, 6606–6610.
(d) H. Onoda, O. Shoji and Y. Watanabe, Dalton Trans., 2015, 44,
7a,b,14
groups,
and we anticipate that the latent potential of
1
5316–15323.
P450BM3 for such reactions could be unlocked by combining
decoy molecules with conventional protein engineering
strategies such as mutagenesis and replacement of haem with
(a) O. Shoji, S. Yanagisawa, J. K. Stanfield, K. Suzuki, Z. Cong, H.
Sugimoto, Y. Shiro and Y. Watanabe, Angew. Chem. Int. Ed., 2017,
5
6, 10324–10329. (b) M. Karasawa, J. K. Stanfield, S. Yanagisawa,
15
artificial metallocomplexes.
O. Shoji and Y. Watanabe, Angew. Chem. Int. Ed., 2018, 57,
12264–12269; Angew. Chem., 2018, 130, 12444-12449.
0 (a) Z. Cong, O. Shoji, C. Kasai, N. Kawakami, H. Sugimoto, Y. Shiro
and Y. Watanabe, ACS Catal., 2015, 5, 150–156. (b) S. Ariyasu, Y.
Kodama, C. Kasai, Z. Cong, J. K. Stanfield, Y. Aiba, Y. Watanabe
and O. Shoji, ChemCatChem, 2019, 11, 4709–4714.
This work was supported by JST CREST Grant Number
JPMJCR15P3 to O.S., Japan. This work was also supported by
JSPS KAKENHI (JP15H05806 to O.S., JP18J23340 to K.S., and
JP18J15250 to Y. S.), Japan.
1
1
1 K. Suzuki, J. K. Stanfield, O. Shoji, S. Yanagisawa, H. Sugimoto, Y.
Shiro and Y. Watanabe, Catal. Sci. Technol., 2017, 7, 3332–3338.
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Stereoselectivity of cyclopropanation of styrene catalysed by cytochrome P450BM3 is enhanced
in the presence of decoy molecules.

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