Biocatalytic route to chiral acyloins: P450-catalyzed regio- and enantioselective α-hydroxylation of ketones

Article abstract of DOI:10.1021/jo502397s

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

Full text of DOI:10.1021/jo502397s

Article
pubs.acs.org/joc
Biocatalytic Route to Chiral Acyloins: P450-Catalyzed Regio- and
Enantioselective α‑Hydroxylation of Ketones
Ruben Agudo,†,‡,§ Gheorghe-Doru Roiban,†,‡,§ Richard Lonsdale,†,‡,§ Adriana Ilie,†,‡,§
́
and Manfred T. Reetz*,†,‡
†Department of Chemistry, Philipps-Universitat Marburg, Hans-Meerwein Strasse, 35032 Marburg, Germany
̈
‡Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany
̈
̈
S
* Supporting Information
ABSTRACT: P450-BM3 and mutants of this monooxygenase
generated by directed evolution are excellent catalysts for the
oxidative α-hydroxylation of ketones with formation of chiral
acyloins with high regioselectivity (up to 99%) and
enantioselectivity (up to 99% ee). This constitutes a new
route to a class of chiral compounds that are useful
intermediates in the synthesis of many kinds of biologically
active compounds.
regio- and enantioselective oxidative α-hydroxylation of ketones
catalyzed by cytochrome P450 monooxygenases (CYPs).12 By
use of 1 as the ketone (Scheme 1), we discovered that wild-type
(WT) P450-BM3 from Bacillus megaterium13 enables the
desired α-hydroxylation with essentially complete regioselec-
tivity (99%) and fairly high enantioselectivity (89% ee) in favor
of (R)-2 being observed (Table 1, entry 1). Ketone 3 reacted
with similar levels of regioselectivity (99%) and enantioselec-
tivity (88% ee); in this case (S)-4 is the favored product (Table 1,
entry 15). These types of acyloins are precursors of various
members of the ephedrine family.
In order to enhance but also to invert stereoselectivity in the
formation of acyloin 2, WT P450-BM3 was subjected to di-
rected evolution14 via structure-guided saturation mutagenesis
at sites around the binding pocket (CASTing).15 Three double-
residue sites [A (V78/T88), B (V78/L181), and C (T268/
A328)] and one single-residue site [D (F87)] surrounding the
catalytic site were mutated (Supporting Information, Figure S1).
The screening effort was reduced by randomizing double-residue
sites with a highly reduced amino acid alphabet15 composed of
only six amino acids (Phe, Tyr, Trp, Lys, Arg, and His) and the
corresponding WT, while NNK (N = adenine/cytosine/
guanine/thymine; K = guanine/thymine) codon degeneracy
was chosen for randomization of site D. In total, 760 trans-
formants were screened, leading to several highly improved
R-selective mutants with up to 99% regio- and 99% enantio-
selectivity (Table 1, entry 6). Only moderately S-selective mutants
were found, namely, F87S and F87T [enantiomeric excess (ee) =
36% and 34%, respectively; Table 1, entries 8 and 9]. In order to
INTRODUCTION
■
Chiral α-hydroxyketones (acyloins) are useful building blocks in
synthetic organic chemistry, and this functionality also occurs in a
number of natural products and therapeutic drugs.1 Due to the
importance of this class of compounds, numerous approaches to
their synthesis have been developed, utilizing a variety of strategies
based on different starting materials, with catalytic asymmetric
processes being of particular interest.1 Prominent methods include
α-hydroxylation of ketones or enolates by use of chiral transition
metal catalysts2 or organocatalysts,3 benzoin condensation with
chiral nucleophilic carbenes as catalysts,4 and monooxidation of
1,2-diols.5 Enzymes as mild and selective catalysts in synthetic
organic chemistry constitute alternative strategies that are often
complementary.6 Several different biocatalytic approaches have
been reported thus far for asymmetric acyloin formation:1
carboligation of aldehydes catalyzed by thiamine diphosphate-
dependent lyases,7 reductase-catalyzed reduction of 1,2-diketones,8
and oxidase-catalyzed oxidation of vicinal diols.9 Moreover,
lipase-catalyzed (dynamic) kinetic resolution of racemic acyloins
and α-acetoxy ketones are also useful entries.10 Finally, chiral
allylic acetates, obtained by lipase-catalyzed dynamic kinetic
resolution, have been subjected to Ru-catalyzed NaIO4-based
oxidation with formation of enantiomerically enriched acyloin
acetates,11a and in one case desymmetrization of 3-phenylpenta-
2,4-dione catalyzed by a Baeyer−Villiger monooxygenase
provided the acylated form of the respective acyloin.11b These
biocatalytic approaches all depend on different starting materials,
which enables flexibility in synthesis planning.
RESULTS AND DISCUSSION
■
In the present study we report a new biocatalytic approach
utilizing completely different starting compounds: namely, the
Received: October 28, 2014
© XXXX American Chemical Society
A
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and WT in the hydroxylation of ketone 3. Only six of these led
to excellent regioselectivity and moderate to good enantio-
selectivity (Table 1, entries 16−21). WT P450-BM3 is highly
selective for formation of (S)-4 (88%). We observe that the
V78W mutant has increased enantioselectivity (92%) over WT
in favor of (S)-4, while A328R displays the highest ee (76%)
with preferential formation of (R)-4.
Scheme 1. Oxidation of Ketones 1 and 3 by P450-BM3
Kinetic parameters TOF (turnover number frequency) and
TTN (total turnover number) were determined for the most
enantioselective mutants used in the formation of 2 and 4,
which show that these mutants are highly active on this type of
substrate (Table 2). The results set the stage for future
bioprocess optimization.
Table 2. Kinetic Parameters for Oxidative Hydroxylation of
a
Ketones 1 and 3
b
c
entry
substrate
P450-BM3
product
TOF
TTN
d
1
2
3
4
5
6
1
1
1
3
3
3
WT
(R)-2
(R)-2
(S)-2
(S)-4
(S)-4
(R)-4
24.1
32.8
16.6
9.5
863
1964
1469
1406
1400
355
F87L
F87T/A328F
d
WT
V78W
A328R
11.4
4.3
a
Catalyzed by the most enantioselective P450-BM3 mutants from
Table 1. Values were obtained by averaging at least three independent
experiments at 75 000 nmol. TOF (turnover number frequency) is
Table 1. Oxidative Hydroxylation of Ketones 1 and 3,
Catalyzed by the Most Regioselective Mutants of
P450-BM3.
b
a
given in nanomoles of product per nanomole of protein per minute.
c
TTN (total turnover number) is given in nanomoles of product per
d
nanomole of protein at the end of reaction. Kinetic parameters of
entry
substrate
P450-BM3
WT
product
% regio
% ee
WT P450-BM3 are indicated for comparison.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
3
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(R)-2
(S)-2
(S)-2
(R)-2
(S)-2
(R)-2
(S)-2
(S)-2
(S)-4
(S)-4
(S)-4
(S)-4
(R)-4
(S)-4
(R)-4
99
98
99
98
98
99
97
98
99
99
89
99
91
91
99
99
99
99
93
99
98
89
94
96
95
6
2
V78F
3
V78H
V78K
In order to expand the substrate scope, we subjected several
substituted propiophenones 5−9 and benzyl methyl ketones
10−14 to oxidative hydroxylation by WT P450-BM3 and some
of the mutants previously evolved for 1. Chart 1 shows that for
most substrates (the exception being 9), both R- and S-selective
mutants were observed. As found for substrate 1, the F87L
mutant is R-enantioselective (93−99%) for reaction with
propiophenones 5−9 but also very S-selective for benzyl
methyl ketones 10 and 11 (94% and 96%, respectively). As is
the case for substrate 3, only low amounts of enantioselectivity
(20−46%) were achieved for R-hydroxylation of 11−14 with
the F87G mutant. Conversely, a very high ee (96%) is
measured for the same mutant with substrate 10.
Docking and MD simulations were performed for 1 with WT
P450-BM3 and the F87T/A328F mutant to rationalize the
observed (opposite) selectivities. Docking calculations alone
were performed for substrate 3 with the WT and A328R
mutant (Supporting Information, Figure S2).
Hydroxylation of aliphatic C−H bonds in P450 enzymes is
widely accepted to occur via the radical rebound mechanism.12
Hence it is assumed that the abstraction of the pro-R and pro-S
hydrogen atoms from 1 will result in formation of (R)-2 and
(S)-2, respectively. The hydrogen atom of the substrate that
spends the largest amount of time in a position close enough to
react with the catalytically active FeVO moiety is used as the
main determinant of selectivity.
4
5
F87A
6
F87L
99
94
36
34
95
6
7
F87R
8
F87S
9
F87T
10
11
12
13
14
15
16
17
18
19
20
21
T88Y
A328F
A328H
F87S/A328F
F87T/A328F
WT
98
94
96
88
89
92
10
34
89
76
V78F
V78W
F87A
F87G
T268 K
A328R
a
Kinetic parameters were measured for the most enantioselective
mutants. Values were obtained by averaging at least three independent
experiments performed at 15 mM.
enhance reversal of enantioselectivity, two different mutants were
constructed by combining mutations from the best S-selective
variants: F87S or F87T with A328F. The resulting variants
showed more than a purely additive16 boost in S-enantio-
selectivity without compromising regioselectivity (94−96% ee;
Table 1, entries 13 and 14).
The shapes of the active-site pockets of the WT and F87T/
A328F mutants promote the formation of (R)-2 and (S)-2,
respectively (Figure 1). The phenyl ring of the substrate forms
a π-stacking interaction with a phenylalanine side chain. This
interaction is formed with F87 in the WT and with F328,
We then tested the 28 mutants that showed measurable
activity toward substrate 1 (Supporting Information, Table S1)
B
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a b
,
Chart 1. Products of α-Hydroxylation of Ketones
a
b
No S-selective mutant was found for 9. Absolute configuration assignment was done by comparing the optical rotation signs with 9−11. Activity of
WT and of the best mutants for all substrates is comparable with TOF numbers for hydroxylation of 1 and 3.
Figure 1. Binding positions of substrate 1 in (a) WT P450-BM3 and (b) F87T/A328F mutant from MD simulations, consistent with formation of
(R)-2 and (S)-2, respectively.
located on the opposite side of the binding pocket, in the
F87T/A328F mutant. In the F87T/A328F mutant, the
carbonyl oxygen of 1 interacts with the hydroxyl group of T87.
Docking poses for substrate 3 in the WT and A328R mutant
are consistent with the observed selectivity (see Supporting
Information). In the A328R mutant, a hydrogen-bonding inter-
action is observed between the carbonyl oxygen of 3 and the
side chain of R328, as well as a π-stacking interaction with F87.
These two interactions put this substrate in a position where
the pro-R hydrogen atom is closest to the heme iron. In the
WT a different binding orientation of 3 is observed in which
the phenyl ring of the substrate forms a closer π-stacking
interaction with F87, the pro-S hydrogen then being closest to
the heme iron.
CONCLUSIONS
■
In summary, we have developed an asymmetric biocatalytic
route to chiral acyloins based on the regio- and enantioselective
P450-BM3-catalyzed oxidative hydroxylation of ketones. The
use of this class of compounds as starting materials for acyloin
synthesis has been reported previously employing chiral
synthetic catalysts2,3 but not biocatalysts such as P450
C
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monooxygenases.17 Thus, the present contribution expands
the toolbox of organic chemists, while widening the substrate
range of P450 enzymes. Structure-based directed evolution14,15
was applied to enhance and to invert enantioselectivity, a
strategy that can be used in future studies when targeting other
ketones.
in TB (400 mL, kan 50 μg/mL) were split in Falcon tubes (25 mL
aliquots) and centrifuged for 7 min at 4000 rpm at room temperature.
The supernatant was discarded and the pellet was resuspended in
5 mL of reaction buffer [phosphate buffer (pH 7.4, 100 mM), glucose
(100 mM), and NADP+ (200 μM)]. Biotransformation was started by
addition of 1 or 3 (15 mM, 75 000 nmol). Reactions were carried out
in an Erlenmeyer flask (100 mL) at 30 °C with mild agitation. Aliquots
(500 μL) were harvested at different time points (20, 60, 120, 180,
240, and 1200 min), extracted with ethyl acetate (500 μL), and
subjected to GC analysis. TOFs were calculated on the basis of
substrate consumption (based on GC results) during the time in which
the highest substrate consumption rate was observed (first 20 min of
reaction). TTNs were calculated at the time point in which maximum
conversion was achieved. All reactions were done in parallel and in
triplicate. Concentration of P450-BM3 variants was measured in
triplicate as previously described.21 Values obtained varied from 2 to
2.5 μM.
Chemistry. General Remarks. Starting compounds were pur-
chased and used without further purification. Phenylacetone was
prepared by a described procedure.22 NMR spectra were recorded on a
300 MHz (1H, 300 MHz; 13C, 75 MHz) spectrometer with
tetramethylsilane (TMS) as internal standard (δ = 0), unless otherwise
noted. Conversion and enantiomeric excess were determined by
achiral and chiral gas chromatography.
EXPERIMENTAL SECTION
■
Biology. General Information. The reagents used have been
described previously elsewhere.18 Escherichia coli BOU730 cells [an E.
coli strain derived from BL21(DE3) that contains a copy of gdh gene
from B. megaterium under the control of T7 promoter]18a were used
during this study. Electrocompetent cells were prepared in-house
according to standard protocols.19
Library Creation. Libraries V78-T88 (library A), V78-L181 (library B),
and T268-A328 (library C) were created by use of a reduced amino
acid alphabet randomization scheme, and library D (F87) was created
by saturation mutagenesis at that position, as previously reported.18b
P450-BM3 Mutant Screening. Screening of the P450-BM3 variants
created by mutagenic PCR was performed by gas chromatography
(GC), as described previously.18 Single BOU730 cell colonies
containing the P450-BM3 variants grown in the presence of
kanamycin (kan, 50 μM) were inoculated into 96-deep-well plates
[each well containing lysogeny broth (LB) (800 μL) and kan (50 μg/mL)]
during 5 h at 37 °C. An aliquot of this culture (100 μL) was added to a
96-deep-well plate [each well containing terrific broth (TB) (900 μL),
kan (50 μg/mL), and isopropyl β-thiogalactoside (IPTG; 0.2 mM)]
and incubated at 30 °C for 20 h. Cells were then centrifuged
(4000 rpm, 15 min at 4 °C), the supernatant was discarded, and the
pellets were resuspended in lysis buffer [each well containing 500 μL
of phosphate buffer (pH 7.4, 100 mM), lysozyme (14 mg/mL), and
DNase I (6 units/mL)]. Plates were incubated for 45 min at 37 °C and
centrifuged at 4000 rpm for 30 min at 4 °C. An aliquot of the resulting
supernatant (350 μL) was transferred to a new 96-deep-well plate
containing 150 μL of reaction buffer [phosphate buffer (pH 7.4,
100 mM), glucose (100 mM), and nicotinamide adenine dinucleotide
phosphate (NADP+, 250 μM)]. Reaction was started by addition of
1 (final concentration 15 mM, 7.5 μmol) and incubated at 25 °C for
20 h. After incubation, samples were extracted with ethyl acetate
(400 μL) and subjected to chiral GC analysis: column Ivadex 1, 25 m;
i.d. 0.25 mm; pressure 1.0 bar H2; injector 230 °C; oven temperature
gradient 50−124 (3 °C/min ramp), 124−220 (25 °C/min ramp);
1 μL; 99:1; FID detector 350 °C.
Small-Scale Reactions. The most enantioselective P450-BM3
mutants for 2 found during the initial screening were sequenced and
grown to perform small-scale reactions. Transformed BOU730 cells
were grown and the best mutants (Table S1, Supporting Information)
were overexpressed in TB (50 mL) as described previously.18 Cell
cultures were split into 2 mL aliquots and centrifuged, and the
resulting pellets were stored at −80 °C for further use. Small-scale
biohydroxylation reactions were performed by resuspending aliquots
in 500 μL of lysis buffer [phosphate buffer (pH 7.4, 100 mM),
lysozyme (14 mg/mL), and DNase I (6 units/mL)] and incubated for
45 min at 37 °C. Reactions were then centrifuged (15 min, 10 000 rpm
at room temperature) and 445 μL of supernatant were transferred to a
new 1.5 mL tube containing 50 μL of glucose (100 mM final
concentration) and 5 μL of NADP+ (250 μM final concentration).
Reaction was started by addition of 1 μL of the corresponding starting
material 1, 3, or 5−14 (9−15 mM, 4.5−7.5 μmol) and incubated at
25 °C (900 rpm) for 20 h. Samples were extracted with ethyl acetate
(500 μL) and subjected to GC analysis by the measuring methods
described below.
Compound Characterization. All reactions were scaled up (100 mg
of starting material approximately) with WT-P450 and P450 mutants, in
order to obtain enough material for NMR characterization (chemical or
bioprocess optimization was not performed). Products were then
purified by column chromatography. They were identified by NMR
spectroscopy and compared with authentic samples as described
previously in the respective literature.
(R)-(+)-2-Hydroxy-1-phenylpropan-1-one 2.
The absolute configuration of (R)-2 was assigned by comparison with
an authentic sample as reported by Luczak and co-workers.23 The
residue was purified by column chromatography (SiO2, ethyl acetate/
1
petroleum ether 1:4). White crystals, 12% yield (13 mg); H NMR
3
3
(300 MHz, CDCl3) δ 7.93 (d, J = 8.2 Hz, 2H), 7.63 (t, J = 7.4 Hz,
1H), 7.51 (t, 3J = 7.7 Hz, 2H), 5.17 (m, 1H), 3.79 (d, 3J = 6.3 Hz, 1H),
1.45 (d, 3J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 202.3, 133.9,
133.3, 128.8 (2C), 128.6 (2C), 69.3, 22.2; [α]2D0 = +58.8 (c 0.83,
CHCl3), lit. (R)-(+)-2 [α]2D0 = +55.4 (c 0.1, CHCl3);23 ee = 99%.
(S)-(+)-1-Hydroxy-1-phenylpropan-2-one 4.
The absolute configuration of (S)-4 was assigned by comparison with
an authentic sample as reported by Schwarz and Meyers.24 The residue
was purified by column chromatography (SiO2, ethyl acetate/
1
petroleum ether 1:4). Colorless liquid, 4% yield (4.4 mg); H NMR
3
(300 MHz, CDCl3) δ 7.29 (m, 5H), 5.03 (d, J = 4.0 Hz, 1H), 4.23
3
(d, J = 4.2 Hz, 1H), 2.02 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 207.1, 138.1, 129.1, 128.8 (2C), 127.5 (2C), 80.3, 25.3; ee = 92%.
(R)-(+)-2-Hydroxy-1-(p-tolyl)propan-1-one 5.
In most cases, small amounts of tautomerized products were
observed, a process that depends on the reaction time and the mutant
used. Partial tautomerization of acyloins is a common phenomenon.20
Turnover Number Determination. Calculation of the kinetic
parameters TOF (turnover number frequency) and TTN (total
turnover number) was done as described previously.18b Cultures of
BOU730 cells overexpressing the corresponding variants of P450-BM3
The absolute configuration of (R)-5 was assigned by comparison with
an authentic sample as reported by Tsuchihashi and co-workers.25 The
residue was purified by column chromatography (SiO2, ethyl acetate/
petroleum ether 1:4). White solid, 23% yield (24.4 mg); H NMR
(300 MHz, CDCl3) δ 7.82 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz,
2H), 5.12 (q, J = 7.0 Hz, 1H), 3.70 (br s, 1H, OH), 2.42 (s, 3H),
1
D
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1.43 (d, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 202.0, 145.1,
130.8, 129.6 (2C), 128.8 (2C), 69.2, 22.5, 21.8; ee = 98%.
(R)-(+)-2-Hydroxy-1-(4-methoxyphenyl)propan-1-one 6.
(300 MHz, CDCl3) δ 7.12 (s, 4H), 4.98 (s, 1H), 3.62 (br s, 1H), 2.27
(s, 3H), 1.99 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 207.4, 138.7,
135.1, 129.8 (2C), 127.4 (2C), 80.0, 25.3, 21.2; ee = 94%.
(S)-(+)-1-Hydroxy-1-(4-methoxyphenyl)propan-2-one 11.
The absolute configuration of (R)-6 was assigned by comparison with
an authentic sample as reported by Luczak and co-workers.23 The
residue was purified by column chromatography (SiO2, ethyl acetate/
The absolute configuration of (S)-11 was assigned by comparison with
an authentic sample as reported by Watson and co-workers.28 The
residue was purified by column chromatography (SiO2, ethyl acetate/
petroleum ether 1:4). Pale yellow crystals, 8% yield (9 mg); 1H NMR
(300 MHz, CDCl3) δ 7.22 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.6 Hz,
2H), 5.04 (s, 1H), 3.80 (s, 3H), 2.06 (s, 3H); ee = 96%.
1
petroleum ether 1:2). Pale yellow oil, 7% yield (8.4 mg); H NMR
(300 MHz, CDCl3) δ 7.91 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz,
2H), 5.10 (q, J = 7.0 Hz, 1H), 3.88 (s, 3H), 3.54 (br s, 1H), 1.44 (d,
J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 200.8, 164.3, 131.1,
126.2, 114.2, 69.0, 55.6, 22.7; ee = 99%.
(S)-(+)-1-(4-Chlorophenyl)-1-hydroxypropan-2-one 12.
(R)-(+)-1-(4-Chlorophenyl)-2-hydroxypropan-1-one 7.
The absolute configuration of (S)-12 was assigned by comparison with
an authentic sample as reported by Yang and co-workers.29 The
residue was purified by column chromatography (SiO2, ethyl acetate/
petroleum ether 1:2). Pale yellow crystals, 29% yield (36.7 mg);
1H NMR (300 MHz, CDCl3) δ 7.30 (d, J = 8.6 Hz, 2H), 7.20 (d, J =
8.6 Hz, 2H), 5.00 (s, 1H), 2.01 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 206.6, 136.6, 134.8, 129.3 (2C), 128.8 (2C), 79.5, 25.3; ee = 85%.
(S)-(+)-1-(4-Bromophenyl)-1-hydroxypropan-2-one 13.
The absolute configuration of (R)-7 was assigned by comparison with
an authentic sample as reported by Muller and co-workers.26 The
̈
residue was purified by column chromatography (SiO2, ethyl acetate/
1
petroleum ether 1:2). Pale yellow crystals, 18% yield (19.7 mg); H
NMR (300 MHz, CDCl3) δ 7.80 (d, J = 8.8 Hz, 2H), 7.40 (d, J =
8.8 Hz, 2H), 5.05 (q, J = 7.0 Hz, 1H), 3.81 (br s, 1H), 1.37 (d, J =
7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 201.3, 140.6, 131.8, 130.1
(2C), 129.3 (2C), 69.4, 22.2; ee = 98%.
(R)-(+)-1-(4-Bromophenyl)-2-hydroxypropan-1-one 8.
The absolute configuration of (S)-13 was assigned by comparing the
optical rotation signs with 10−12. The residue was purified by column
chromatography (SiO2, ethyl acetate/petroleum ether 1:4). Pale
The absolute configuration of (R)-8 was assigned by comparison with
an authentic sample as reported by Muller and co-workers.26 The
̈
1
yellow crystals, 56% yield (60 mg); H NMR (300 MHz, CDCl3)
residue was purified by column chromatography (SiO2, ethyl acetate/
δ 7.52 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 5.05 (s, 1H), 2.08
(s, 3H); 13C NMR (75 MHz, CDCl3) δ 206.5, 137.1, 132.3 (2C),
129.1 (2C), 122.9, 79.6, 25.3; ee = 69%.
1
petroleum ether 1:2). Colorless crystals, 4% yield (4 mg); H NMR
(300 MHz, CDCl3) δ 7.73 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz,
2H), 5.04 (q, J = 7.0 Hz, 1H), 3.65 (br s, 1H), 1.37 (d, J = 7.0 Hz,
3H); 13C NMR (75 MHz, CDCl3) δ 201.5, 132.4 (2C), 132.2, 130.2
(2C), 129.3, 69.4, 22.3; ee = 93%.
(S)-(+)-1-(2-Fluorophenyl)-1-hydroxypropan-2-one 14.
(R)-(+)-1-(2-Fluorophenyl)-2-hydroxypropan-1-one 9.
The absolute configuration of (S)-14 was assigned by comparison with
an authentic sample as reported by Turner and co-workers.30 The
residue was purified by column chromatography (SiO2, ethyl acetate/
petroleum ether 1:5). Pale yellow viscous oil, 43% yield (48 mg);
1H NMR (300 MHz, CDCl3) δ 7.31−7.18 (m, 2H), 7.14−6.97
(m, 2H), 5.34 (s, 1H), 3.88 (br s, 1H), 2.05 (d, J = 0.7 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 205.1 (d, J = 1.2 Hz, 1C), 159.3 (d, J =
247.1 Hz, 1C), 129.3 (d, J = 8.3 Hz, 1C), 127.7 (d, J = 3.6 Hz, 1C),
124.2 (d, J = 13.6 Hz, 1C), 123.7 (d, J = 3.6 Hz, 1C), 114.8 (d, J = 21.6
Hz, 1C), 72.6 (d, J = 3.4 Hz, 1C), 23.9 (d, J = 2.3 Hz, 1C); ee = 65%.
Molecular Docking Calculations: Details of Docking and
Molecular Dynamics Simulations. Docking calculations were
performed with the AUTODOCK VINA31 program within the
PyMol interface.32 The structures of substrates 1 and 2 were built in
the Maestro program,33 and a geometry optimization was performed
by use of the ORCA program (version 3.0.1) at the BP86/SV level.34
Preliminary simulations were performed starting from the 1BU7
crystal structure. However, in this structure the substrate (1) failed to
remain in the active site. The 1JPZ crystal structure35 contains a
cocrystallized ligand (N-palmitoylglycine) and hence has a larger active
site cavity than the 1BU7 structure. This ligand was removed prior to
docking and MD simulations. When the 1JPZ crystal structure was
used, the substrate remained in the active site for the entirety of the
simulations. Mutations were performed within PyMol, and the
The absolute configuration of (R)-9 was assigned by comparison with
an authentic sample as reported by Muller and co-workers.26 The
̈
residue was purified by column chromatography (SiO2, ethyl acetate/
1
petroleum ether 1:4). Pale yellow viscous oil, 24% yield (30 mg); H
NMR (300 MHz, CDCl3) δ 7.95−7.78 (m, 1H), 7.56−7.48 (m, 1H),
7.22 (td, J = 7.8, 1.0 Hz, 1H), 7.13−7.06 (m, 1H), 4.98 (qd, J =
7.0, 2.8 Hz, 1H), 3.58 (br s, 1H, OH), 1.33 (dd, J = 7.0, 1.5 Hz, 3H);
13C NMR (75 MHz, CDCl3) δ 201.0 (d, J = 4.4 Hz, 1C), 161.7 (d, J =
255.4 Hz, 1C), 135.6 (d, J = 9.2 Hz, 1C), 131.23 (d, J = 2.8 Hz, 1C),
124.9 (d, J = 3.3 Hz, 1C), 122.3 (d, J = 13.4 Hz, 1C), 116.8 (d, J =
23.6 Hz, 1C), 72.8 (d, J = 9.2 Hz, 1C), 20.8 (d, J = 1.1 Hz, 1C);
ee = 97%.
(S)-(+)-1-Hydroxy-1-(p-tolyl)propan-2-one 10.
The absolute configuration of (S)-10 was assigned by comparison with
an authentic sample as reported by Fleming et al.27 The residue was
purified by column chromatography (SiO2, ethyl acetate/petroleum
ether 1:4). Pale yellow crystals, 26% yield (28.3 mg); 1H NMR
E
dx.doi.org/10.1021/jo502397s | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
conformations of mutated residues were selected such to minimize
steric clashes.
(4) (a) Enders, D.; Kallfass, U. Angew. Chem., Int. Ed. 2002, 41,
1743−1745. (b) Marti, J.; Castells, J.; Lopez-Calahorra, F. Tetrahedron
Lett. 1993, 34, 521−524.
For each MD simulation, the protein was solvated and neutralized
with the tleap program in a truncated octahedron water box of
sufficient size to provide a solvent layer of a minimum of 12 Å thick-
ness surrounding the protein. The simulation system was neutralized
by the addition of Na+ ions. Molecular dynamics simulations were
performed with the Amber12 program36with the FF12SB molecular
mechanics force field37 and TIP4P-EW water model.38 Molecular
mechanics parameters for the cysteine-coordinated heme in the
Compound I state were obtained from ref 39. The parameters and
topology of substrate 1 were obtained by use of the ANTECHAMBER
program with the generalized Amber force field (GAFF).
The minimization and equilibration procedure for the relaxation of
each system prior to production-phase simulations was the same as
described previously.40 Two unrestrained 24 ns NPT simulations were
performed on the selected docking poses for both the WT and F87T/
A328F mutant.
(5) (a) Zhang, J. D.; Xu, T. T.; Li, Z. Adv. Synth. Catal. 2013, 355,
3147−3153. (b) Onomura, O.; Arimoto, H.; Matsumura, Y.; Demizu,
Y. Tetrahedron Lett. 2007, 48, 8668−8672.
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Organic Chemistry, 3rd ed.; Wiley−VCH: Weinheim, Germany, 2012.
(b) Perspective on enzymes in organic chemistry: Reetz, M. T. J. Am.
Chem. Soc. 2013, 135, 12480−12496.
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2904. (b) Hischer, T.; Gocke, D.; Fernandez, M.; Hoyos, P.; Alcantara,
A. R.; Sinisterra, J. V.; Hartmeier, W.; Ansorge-Schumacher, M. B.
Tetrahedron 2005, 61, 7378−7383. (c) Dominguez de Maria, P.;
Stillger, T.; Pohl, M.; Kiesel, M.; Liese, A.; Groger, H.; Trauthwein, H.
̈
Adv. Synth. Catal. 2008, 350, 165−173. (d) Westphal, R.; Vogel, C.;
Schmitz, C.; Pleiss, J.; Muller, M.; Pohl, M.; Rother, D. Angew. Chem.,
̈
Int. Ed. 2014, 53, 9376−9379.
For the F87T/A328F mutant, two 12 ns simulations were first
performed on the substrate-free mutant prior to docking, in order to
allow the protein to relax in the presence of the mutated residues. The
structures from the two simulations were then clustered in the cpptraj
program. Clustering was performed on the basis of the positions of
residues lining the active-site cavity (residues 75, 78, 82, 87, 88, 177,
181, 260, 263, 264, 267, 268, 328, 437, and 438). Six clusters were
obtained by use of the average-linkage algorithm. Prior to clustering, all
water molecules were removed. The docking of substrate 1 was then
performed on the most populated representative structure from the
clustering calculation. Following the selection of a docking pose, two
24 ns MD simulations were performed, repeating the solvation,
minimization, and equilibration steps as detailed above.
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71, 7632−7637. (c) Hoyos, P.; Dominguez de Maria, P.; Sinisterra, J.
V.; Alcantara, A. R. Hydrolase based synthesis of enantiopure R-hydroxy
ketones: From racemic resolutions to chemo-enzymatic dynamic kinetic
resolutions. ; Biotechnology Research: Technology and Applications;
Nova Science Publishers, Inc.: New York, 2009; pp 97−119.
(d) Tanyeli, C.; Akhmedov, I.; Iyigun, C. Tetrahedron: Asymmetry
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text describing detailed results from docking and
MD simulations; one table listing P450-BM3 hits found after
screening with 1; seven figures showing randomization sites
A−D, docking results for 3, and RMSD of backbone heavy
atoms and distances calculated during MD simulations of 1;
selected NMR spectra and GC chromatograms. This material is
available free of charge via the Internet at http://pubs.acs.org/.
̈
2004, 15, 2861−2869. (f) Odman, P.; Wessjohann, L. A.;
Bornscheuer, U. T. J. Org. Chem. 2005, 70, 9551−9555. (g) Petrenz,
A.; Dominguez de Maria, P.; Ramanathan, A.; Hanefeld, U.; Ansorge-
Schumacher, M. B.; Kara, S. J. Mol. Catal. B: Enzym. 2015, 396, 328−
334.
AUTHOR INFORMATION
Corresponding Author
*E-mail reetz@mpi-muelheim.mpg.de.
■
(11) (a) Bogar, K.; Hoyos Vidal, P.; Alcantara Leon, A. R.; Backvall,
̈
J.-E. Org. Lett. 2007, 9, 3401−3404. (b) de Gonzalo, G.; Torress
Pazmino, D. E.; Ottolina, G.; Fraaije, M. W.; Carrea, G. Tetrahedron:
Asymmetry 2005, 16, 3077−3083.
Author Contributions
§R.A., G.-D.R., R.L., and A.I. contributed equally.
(12) Reviews of P450 monooxygenases: (a) Ortiz de Montellano, P.
R. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.;
Springer: Berlin, 2005. (b) Isin, E. M.; Guengerich, F. P. Biochim.
Biophys. Acta, Gen. Subj. 2007, 1770, 314−329. (c) Ortiz de
Montellano, P. R. Chem. Rev. 2010, 110, 932−948. (d) Whitehouse,
C. J. C.; Bell, S. G.; Wong, L.-L. Chem. Soc. Rev. 2012, 41, 1218−1260.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support by the Max-Planck-Society and the Arthur C.
Cope Foundation is gratefully acknowledged.
■
(e) O’Reilly, E.; Kohler, V.; Flitsch, S. L.; Turner, N. J. Chem. Commun.
̈
2011, 47, 2490−2501. (f) Fasan, R. ACS Catal. 2012, 2, 647−666.
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Ringle, M.; Janocha, S.; Leize-Wagner, E.; Urlacher, V. B.; Bernhardt,
R. Biotechnol. Appl. Biochem. 2013, 60, 18−29. (h) Holtmann, D.;
Fraaije, M. W.; Arends, I. W. C. E.; Opperman, D. J.; Hollmann, F.
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