Regio- and Stereoselective Oxidation of Styrene Derivatives to Arylalkanoic Acids via One-Pot Cascade Biotransformations

Article abstract of DOI:10.1002/adsc.201700416

Green and selective oxidation methods are highly desired in chemical synthesis and manufacturing. In this work, we have developed a biocatalytic method for the regio- and stereoselective oxidation of styrene derivatives into arylacetic and (S)-2-arylpropionic acids via a one-pot epoxidation–isomerization–oxidation sequence. This was done via the engineering of Escherichia coli (StyABC-EcALDH) coexpressing styrene monooxygenase (SMO), styrene oxide isomerase (SOI) and aldehyde dehydrogenase (EcALDH) as an active and easily available whole-cell catalyst. Regioselective oxidation of styrene and 11 substituted styrenes using the E. coli cells was performed in a one-pot set-up, producing 12 phenylacetic acids in both high conversion and high yield. Engineering of E. coli (StyABC-ADH9v1) coexpressing SMO, SOI and ADH9v1 (a mutated alcohol dehydrogenase) led to biocatalysts capable of regio- and stereoselective oxidation of α-methylstyrene derivatives to the corresponding chiral acids. One-pot asymmetric synthesis of 4 (S)-2-arylpropionic acids was achieved in good conversion and excellent ee with the E. coli cells. This is a new type of asymmetric alkene oxidation to give chiral acids with no chemical counterpart thus far. The cascade bio-oxidation operates under mild conditions, uses molecular oxygen, exhibits very high regio- and enantioselectivity, and gives high conversion, thus providing a green and efficient method for the synthesis of arylacetic acids and (S)-2-arylpropionic acids directly from easily available styrenes. (Figure presented.).

Full text of DOI:10.1002/adsc.201700416

Title: Regio- and Stereoselective Oxidation of Styrene Derivatives to
Aryl Acids via One-Pot Cascade Biotransformations
Authors: Shuke Wu, Yi Zhou, Daniel Seet, and Zhi Li
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201700416
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Regio- and Stereoselective Oxidation of Styrene Derivatives to
Aryl Acids via One-Pot Cascade Biotransformations
Shuke Wu,a, b Yi Zhou,b Daniel Seet,a and Zhi Lia, b,*
a
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4,
Singapore 117585
Fax: (+65)-6779-1936; phone: (+65)-6516-8416; e-mail: chelz@nus.edu.sg
Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of
b
Singapore, 28 Medical Drive, Singapore 117456
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Green and selective oxidation methods are highly and stereoselective oxidation of α-methylstyrene derivatives
desired in chemical synthesis and manufacturing. In this to the corresponding chiral acids. One-pot asymmetric
paper, we developed a biocatalytic method for regio- and synthesis of 4 (S)-2-arylpropionic acids was achieved in
stereoselective oxidation of styrene derivatives into good conversion and excellent ee with the E. coli cells. This
arylacetic and (S)-2-arylpropionic acids via one-pot is a new type of asymmetric alkene oxidation to give chiral
epoxidation-isomerization-oxidation. This was done via the acids with no chemical counterpart thus far. The cascade
engineering of Escherichia coli (StyABC-EcALDH) bio-oxidation operates in mild conditions, uses molecular
coexpressing styrene monooxygenase (SMO), styrene oxide oxygen, exhibits very high regio- and enantioselectivity,
isomerase (SOI) and aldehyde dehydrogenase (EcALDH) as and gives high conversion, thus providing a green and
an active and easily available whole-cell catalyst. efficient method for the synthesis of arylacetic acids and
Regioselective oxidation of styrene and 11 substituted (S)-2-arylpropionic acids directly from easily available
styrenes using the E. coli cells was performed in a one-pot styrenes.
setup, producing 12 phenylacetic acids in both high
conversion and high yield. Engineering of E. coli (StyABC-
ADH9v1) coexpressing SMO, SOI and ADH9v1 (a mutated
alcohol dehydrogenase) led to biocatalysts capable of regio-
Keywords: Alkenes; Biotransformations; Cascade
reactions; Enzyme catalysis; Oxidation
such as diols,[8b,8d,8e] α-hydroxy acids,[8g] amino
alcohols,[8g] and α-amino acids.[8g] Recently, we have
been interested in developing a green and efficient
oxidation method to convert alkenes to the
corresponding (chiral) terminal acids (Scheme 1).
Introduction
Performing oxidation reactions in a selective and
environmentally benign manner is very important in
the chemical industry for transforming highly
reduced hydrocarbons (major feedstock) to various
useful oxygenated products. Enzyme catalysis has
become a green method for organic synthesis, due to
the high chemo- regio- and enantioselectivity and
mild reaction conditions.[1] Notably, enzymatic
oxidation often uses green and cheap O2 as the
oxidant with no toxic or precious metals.[2] In
addition to single-step bio-oxidations,[3] enzymatic
oxidation could be combined with other enzymatic
reactions to achieve enzyme cascades for new types
of useful oxidations and syntheses.[4] For instance,
alcohol oxidation,[5] hydroxylation,[6] and Baeyer–
Villiger oxidation[7] were employed in cascades to
enable various oxidative transformations. We
previously developed several new oxidative
cascades[8] for one-pot functionalization of alkenes to
produce many oxygen-containing chiral compounds,
Scheme 1. Cascade biocatalysis for functionalization of
styrenes to diverse types of compounds.
1
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
Scheme 2. Cascade biotransformation of styrene derivatives to acids via epoxidation-isomerization-oxidation. a) from
styrenes to arylacetic acids with E. coli (StyABC-EcALDH) cells; b) from 2-arylpropenes to (S)-2-arylpropionic acids
with E. coli (StyABC-ADH9v1) cells. SMO: styrene monooxygenase; SOI: styrene oxide isomerase; EcALDH: an
aldehyde dehydrogenase from E. coli; ADH9v1: a mutated alcohol dehydrogenase from Pantoea.
Alkenes are easily available hydrocarbons for
chemical synthesis. Regioselective oxidation of
styrenes to arylacetic acids was recently reported with
iodine catalyst and oxone.[9] However, oxone is
suffered from low atom efficiency, the catalytic
turnover of iodine is low, and the system is unable to
convert 1,1-disubstituted alkenes into chiral acids.
We have been interested in developing a biocatalytic
system for this oxidation by retrofitting the styrene
monooxygenase (SMO)[10] and styrene oxide
isomerase (SOI)[11] in the styrene degradation
pathway with another aldehyde dehydrogenase
(ALDH).[12] The conversion of styrene to
phenylacetic acid was observed in some styrene-
degrading wild-type strains,[13] but it has not been
demonstrated for synthetic applications.
acids, and the application of the biocatalysts for
synthesis of 12 useful arylacetic acids in high
conversion and yield and
4
valuable (S)-2-
arylpropionic acids in high conversion and excellent
ee.
Results and Discussion
Genetic Engineering and Characterization of E.
coli (StyABC-EcALDH)
To engineer the desired cascade, SMO and SOI in the
styrene degradation pathway were selected for the
epoxidation and isomerization, respectively (Scheme
2). For the oxidation of phenylacetaldehyde,
EcALDH from E. coli was reported to have high
activity.[12] We started with the engineering of
recombinant E. coli expressing SMO, SOI, and
EcALDH, as whole-cell biocatalysts for cascade
biotransformation of 1 to 4, due to the following
reasons: 1) whole cells provide a natural environment
for high activity of the flavin-depended SMO and the
membrane-integrated SOI;[17] 2) heterogeneous
expression in E. coli instead of natural host strain
could avoid the further degradation of phenylacetic
acid products; 3) recombinant E. coli can be easily
cultured to high cell density using glucose in simple
medium. We previously cloned the SMO genes on
pRSF-StyAB.[8e] The gene of SOI (styC) was
synthesized and amplified, while the gene of
EcALDH was amplified from previously constructed
plasmid pRSF-AlkJ-EcALDH.[8g] The gene fragments
of styC and aldh were constructed together with the
pRSF-StyAB plasmid at the same time to form a new
A group of substituted styrenes 1a-1p were
selected as the model substrates for the one-pot
oxidation to synthesize arylacetic acids 4a-4l and (S)-
2-arylpropanoic acids 4m-4p, respectively (Scheme
2). Phenylacetic acid 4a and its esters are widely
applied as flavors and fragrances.[14a] Substituted
phenylacetic acids are commonly used intermediates
to develop new reactions[14b-d] and synthesize many
potent
bioactive
compounds.[14e-g]
(S)-2-
Arylpropanoic acids (S)-4m-4n are key chiral
synthons of several bioactive reagents and chiral
organocatalysts.[15] Several bulky (S)-2-arylpropanoic
acids are the active enantiomers of several
nonsteroidal anti-inflammatory drugs (NSAIDs, e.g.
ibuprofen, naproxen, and flurbiprofen).[16]
Here, we report the engineering of E. coli strains
expressing SMO, SOI, and EcALDH or ADH9v1 as
active and easily available whole-cell catalysts for
regio- and stereoselective oxidation of alkenes to
2
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
Figure 1. a) Genetic construction of plasmid pRSF-
StyABC-EcALDH; b) genetic construction of plasmid
pRSF-StyABC-ADH9v1; c) SDS-PAGE analysis of cell
protein of E. coli (StyABC-EcALDH) (lane 1) and E. coli
(StyABC-ADH9v1) (lane 2).
Figure 2. Optimization of reaction conditions of 1a (150
mM) to 4a using E. coli (StyABC-EcALDH) resting cells
(10 g cdw L–1) at 30 °C. a) biotransformation in a two-
liquid phase system of KP buffer (200 mM, pH 8.0, 0.5%
glucose) and different organic phase (1:1); HA: hexyl
acetate, OA: octyl acetate, EO: ethyl oleate, C6: n-hexane,
recombinant
plasmid
pRSF-StyABC-EcALDH
(Figure 1a). This plasmid was transformed into E.
coli T7 express strain to give E. coli (StyABC-
EcALDH).
C12:
n-dodecane,
C16:
n-hexadecane;
b)
The engineered E. coli strain grew well in M9
medium supplemented with glucose and yeast extract,
and the expression of SMO, SOI, and EcALDH was
induced by adding IPTG. After an overnight (12-13
h) culture at 22 °C, the cells were harvested for
biotransformation in EO and KP buffer (400 mM, pH 8.0)
containing different amounts of glucose.
(potassium phosphate) buffer as the reaction medium.
Long chain ester, ethyl oleate (EO), and long chain
alkane, n-hexadecane, were found to be the best
(Figure 2a).
characterization
and
biotransformation.
The
expression of StyA, StyB, StyC, and EcALDH was
clearly observed on SDS-PAGE analysis of the cell
protein of E. coli (StyABC-EcALDH) (lane 1 in
Figure 1c). The activity of each enzyme in the cells of
E. coli (StyABC-EcALDH) was measured: SMO
activity is 56 U (g cdw)–1 for epoxidation of 1a; SOI
activity is 12 kU (g cdw)–1 for isomerization of 2a;
and EcALDH activity is 81 U (g cdw)–1 for oxidation
of 3a. Clearly, the second and third reaction steps are
faster than the first reaction step, which will lead to a
Theoretically, the cascade enables the recycling of
NAD+/NADH cofactors inside of E. coli cells. As
SMO may require slightly more NADH due to the
auto-oxidation of FADH2, some glucose may be
required for the regeneration of NADH for this.
However, adding too much glucose may influence the
third step, aldehyde oxidation using NAD+. Based on
experiment, a small amount of glucose (0.5% w/v)
gave the best biotransformation result (Figure 2b).
We also optimized the KP buffer, temperature, and
the volume ratio of KP buffer to EO (Figure S1 in
supporting information). As expected, the best
condition is KP buffer (pH 8.0, 400 mM) and 30 °C,
while the ratio of KP buffer: EO = 1–5: 1 did not
significantly influence the biotransformation. To
reduce the use of EO, the ratio of KP buffer to EO =
5: 1 is applied in the preparative biotransformation.
clean
cascade
transformation
without
the
accumulation of intermediates during the reaction.
The apparent Kd value of FAD binding to StyA (the
oxygenase component of SMO) was 21.2 μM,[18a]
while FAD concentration in E. coli was 0.17 mM,[18b]
which is much higher than the Kd value. Thus, E. coli
could provide enough FAD for a proper activity of
SMO, and kinetic modeling of the cascade may help
to reveal the bottleneck.[18c]
Optimization of Reaction Conditions of Cascade
Biotransformation of 1a to 4a
Time Course of Cascade Biotransformation of 1a
to 4a in High Concentration
The E. coli (StyABC-EcALDH) resting cells (10 g
It is very important to study the detailed time course
of cascade reactions to investigate the accumulation
of intermediate and formation of by-products. A
maximum of ~80% conversion to 4a was achieved
from 150 mM 1a during the optimization experiment,
thus a slightly lower concentration of 1a (130 mM)
–
1
cdw L ) were examined for the conversion of 1a (150
mM) to 4a under different reaction conditions in
small scale. Since styrene 1a is highly hydrophobic
and toxic to cells, different organic solvents were
explored to form two-liquid phase systems with KP
3
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
Table 1. One-pot synthesis of arylacetic acids 4a-4l from
styrenes 1a-1l with E. coli (StyABC-EcALDH).[a]
Sub.
X
Conc. Prod. Conv. to Isolated
[mM]
100
50
4 [%][b]
96
yield [%]
H
82
1a
1b
1c
1d
1e
1f
4a
4b
4c
4d
4e
4f
o-F
98
71
m-F
50
>99
98
73
p-F
50
76
m-Cl
p-Cl
m-Br
p-Br
m-Me
p-Me
m-OMe
p-OMe
50
94
81
50
85
75
25
91
77
1g
1h
1i
4g
4h
4i
25
87
71
Figure 3. Time curve of biotransformation of 130 mM 1a
to 4a with E. coli (StyABC-EcALDH) resting cells (15 g
cdw L–1) in a two-liquid phase system of KP buffer (400
mM, pH 8.0, 0.5% glucose) and EO (1:1) at 30 °C. 2a and
3a were not observed.
50
99
67
50
98
56
1j
4j
50
>99
>99
66
1k
4k
4l
50
52
1l
[a]
Biotransformation of 1b-1l was performed with E. coli
(StyABC-EcALDH) resting cells (15 g cdw L–1) in a two-
liquid phase system of 40 mL KP buffer (400 mM, pH 8.0,
0.5% glucose) and 8 mL EO at 30 °C for 10 h. Reaction of
was applied to ensure the high conversion to 4a.
Time course of cascade biotransformation of 1a (130
mM) with E. coli (StyABC-EcALDH) resting cells
–
1
1a was conducted in the same conditions but larger volume
of 100 mL KP buffer and 20 mL EO.
HPLC analysis.
(15 g cdw L ) was shown in Figure 3. 1a was quickly
converted into 4a without the accumulation of
intermediate 2a or 3a. At the end of reaction (6 h),
122 mM of 4a (16.6 g L–1) was produced,
corresponding to 94% conversion from 1a. While 5
mM of 1a remained in the system, no other by-
product was detectable. Clearly, this three-step
cascade proceeds as a single-step oxidation of styrene
to phenylacetic acid with O2.
[b]
Determined by
and flash chromatography, afforded 153-276 mg of
4b-4l with 52-81% isolated yield. These results
confirmed the synthetic utility of the epoxidation-
isomerization-oxidation cascade for the one-pot
oxidation of styrene derivatives to prepare many
useful phenylacetic acids.
It was reported that phenylacetic acids could be
produced via regioselective oxidation of styrenes
using iodine catalyst with oxone as terminal
oxidant,[9] but the catalytic turnover was low and the
oxidant was low in atom efficiency. In comparison,
our biocatalytic system uses whole-cell biocatalysts
with molecular oxygen as the oxidant, thus it could
be greener.
One-Pot Synthesis of Arylacetic Acids 4a-4l from
Styrenes 1a-1l with E. coli (StyABC-EcALDH)
To demonstrate the synthetic utility of the cascade,
we applied E. coli (StyABC-EcALDH) resting cells
–
1
(15 g cdw L ) to transform 1a (100 mM) in a larger
system (100 mL KP buffer and 20 mL EO) under the
optimal reaction conditions. After 10 hour reaction,
4a was produced in 96%. The reaction mixture was
subjected to centrifugation to remove the cells and
EO. The aqueous solution was acidified with HCl,
saturated with NaCl, and extracted with ethyl acetate.
Then, the ethyl acetate was evaporated and the crude
product was purified with flash chromatography.
Finally, pure 4a (1116 mg) was obtained in 82%
isolated yield (Table 1).
Genetic Engineering and Characterization of E.
coli (StyABC-ADH9v1)
Enzymes often possess high stereoselectivity for
various reactions, thus being a valuable tool for
asymmetric synthesis. To explore the biocatalytic
cascade for enantioselective oxidation of alkenes to
chiral acids, we tested E. coli (StyABC-EcALDH) for
oxidation of α-methylstyrene. Biotransformation of
1m (10 mM) with E. coli (StyABC-EcALDH) (10 g
cdw L–1) gave (S)-2-phenylpropanoic acid (S)-4m in
96% conversion. However, the ee of (S)-4m was only
88%. A possible reason could be that the intermediate
Since many substituted phenylacetic acids are also
useful fine chemicals with their niche applications,
the cascade biotransformation was implemented to
prepare 4b-4l in 100-200 mg scale. Biotransformation
of 1b-1l (25-50 mM) was performed with E. coli
–
1
(StyABC-EcALDH) resting cells (15 g cdw L ) in a
system containing 40 mL KP buffer and 8 mL EO.
The conversion to 4 was determined to be 85->99%
after 10 hours (Table 1). Similar work-up, extraction,
(S)-3m encountered racemization in the aqueous
20]
buffer[19,
and EcALDH is not enantioselective
4
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
Table 2. One-pot asymmetric synthesis of (S)-2-
arylpropionic acids (S)-4m-4p from α-methylstyrene
derivatives 1m-1p with E. coli (StyABC-ADH9v1).[a]
enough for oxidation of 3m to give (S)-4m in high ee.
Without further oxidation to (S)-4m, racemization of
(S)-3m was observed in the two-phase system (Figure
S2 in supporting information).
To produce (S)-4m in very high ee, we cloned
alcohol dehydrogenase ADH9v1 which was reported
with S-selectivity for enantioselective oxidation of
racemic 3m.[20] The gene of ADH9v1 was
synthesized and constructed together with styC and
pRSF-StyAB to form a recombinant plasmid pRSF-
StyABC-ADH9v1 (Figure 1b). This plasmid was
transformed into E. coli T7 express strain to give E.
coli (StyABC-ADH9v1). The strain was grown and
induced under the same cultivation procedure for E.
coli (StyABC-EcALDH). SDS-PAGE analysis of the
cell protein confirmed the expression of StyA, StyB,
StyC, and ADH9v1 (lane 2 in Figure 1c).
Su
b.
X
Conc.
[mM]
Prod.
Conv. ee
Isol.
to
4
[%][b] yield
[%]
[%][b]
H
20
5
5
(S)-4m
(S)-4n
(S)-4o
(S)-4p
82
75
67
73
98
97
92
98
65
49
46
52
1m
1n
1o
p-F
p-Cl
p-Me
5
1p
[a]
Biotransformation of 1m-1p was performed with E. coli
(StyABC-ADH9v1) resting cells (20 g cdw L–1) in a two-
liquid phase system of 100 mL KP buffer (200 mM, pH
One-Pot Synthesis of (S)-Arylpropionic Acids 4m-
4p from α-Methylstyrene Derivatives 1m-1p with
E. coli (StyABC-ADH9v1)
[b]
8.0) and 10 mL n-hexadecane at 30 °C for 24 h.
Determined by chiral HPLC analysis.
Asymmetric cascade oxidation of 1m (20 mM) was
examined with resting cells of E. coli (StyABC-
ADH9v1) under different conditions in small scale
(Figure S3 in supporting information). The ee of (S)-
4m was excellent (98-99%) under all these conditions,
and high conversion (80%) was achieved with resting
enantioselective enzymes may be developed by
directed evolution[21] and balanced expression of
enzymes could be achieved by engineering of
promoters or ribosome-binding sites.[22]
–
1
cells (20 g cdw L ) and no glucose.
Conclusion
To demonstrate the cascade oxidation for
asymmetric synthesis of 2-arylpropionic acid, E. coli
–
A green and efficient synthetic method for regio- and
stereoselective conversion of terminal alkenes to
terminal acids was developed via one-pot
epoxidation-isomerization-oxidation. Active and
easily producible recombinant E. coli whole-cell
biocatalysts were engineered by retrofitting styrene
degradation enzymes, SMO and SOI, with a selected
aldehyde dehydrogenase. Cascade oxidation of
styrene and 11 substituted styrenes using E. coli
(StyABC-EcALDH) gave rise to a facile synthesis of
12 useful aryl acids in high conversion and high yield.
The one-pot biosynthetic method could be greener
and more efficient than the corresponding only
known chemical method. More importantly, highly
regio- and stereoselective oxidation of α-
methylstyrene derivatives to chiral aryl acids was also
demonstrated. E. coli (StyABC-ADH9v1) containing
three enantioselective enzymes (SMO, SOI,
ADH9v1) was engineered, which catalyzed the
asymmetric oxidation of four α-methylstyrene
derivatives to the corresponding useful and valuable
(S)-2-arylpropionic acids in excellent ee with good
conversion. To the best of our knowledge, this is the
first asymmetric oxidation of alkenes to chiral acids.
The cascade reaction operates under mild conditions,
utilizes non-toxic enzymes, consumes O2 as the
stoichiometric oxidant, and enables intracellular
cofactor recycling, thus being a green and efficient
method for direct synthesis of terminal acids from
terminal alkenes. Further exploring the cascade
reaction for the synthesis of (S)-NSAIDs drugs is
under way.
1
(StyABC-ADH9v1) resting cells (20 g cdw L ) were
employed to transform 1m (20 mM) in a larger
system consisting of 100 mL KP buffer and 10 mL n-
hexadecane. After reacting for 24 hours, (S)-4m was
produced in 82% conversion (Table 2). Work-up,
extraction with ethyl acetate, and purification by flash
chromatography gave 195 mg of pure (S)-4m in 65%
isolated yield. The ee of (S)-4m is excellent (98%).
The cascade bio-oxidation was further applied to
transform ring-substituted α-methylstyrenes 1n-1p (5
mM) in the same system of 100 mL KP buffer and 10
mL n-hexadecane. (S)-4n-4p were successfully
produced in 67-75% conversion, and similar work-up,
extraction, and purification afforded pure (S)-4n-4p
in 46-52% isolated yield. The ee of (S)-4n and (S)-4p
was also excellent (97-98%), while the ee of (S)-4o is
slightly lower (92%). These results clearly
demonstrated
that
epoxidation-isomerization-
oxidation cascade is highly regio- and stereoselective
for the conversion of 2-arylpropenes to give (S)-2-
arylpropionic acids. This unique one-pot asymmetric
oxidation has no chemical counterpart thus far. The
iodine catalyst with oxone was unsuccessful for the
enantioselective oxidation.[9]
In principle, this cascade reaction is applicable for
the oxidation of 2-arylpropene to produce (S)-2-
arylpropionoic acids that are the active enantiomers
of several NSAIDs (ibuprofen, naproxen, and
flurbiprofen).[16] We are currently working on
adapting our cascade oxidation system for such
synthesis. If necessary, more active or
5
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
Experimental Section
(S)-4n 6.3 min, (R)-4o 6.0 min, (S)-4o 6.6 min, (R)-4p 5.7
min, (S)-4p 6.4 min.
Chemicals
Culture of E. coli Strains for Biotransformation
The following chemicals were purchased from Sigma-
Aldrich: 1a-1k, 1m, 1p, 2a, 3a, 4a-4l, rac-4m-4p, (S)-4m,
acetic acid, ethyl oleate, n-hexadecane, kanamycin,
glucose, NaCl, Na2SO4, Na2HPO4, NH4Cl, KH2PO4,
K2HPO4, TFA, phenethylamine, and benzyl alcohol. 1l was
from Alfa Asear. 1n and 1o were from TCI chemical.
Acetonitrile, ethyl acetate, isopropanol and n-hexane were
purchased from Tedia. n-Heptane, silica gel 60 and TLC
plates were purchased from Merck. LB medium, yeast
extract, and agar were purchased from Biomed Diagnostics.
DNA polymerase, ligase, and restriction enzymes were
purchased from Thermo Fisher.
E. coli (StyABC-EcALDH) or E. coli (StyABC-ADH9v1)
was initially inoculated in LB medium (1 mL) with 50 mg
L– 1 kanamycin and grew for 8-10 h at 37 °C. This culture
was then transferred to 50 mL M9 medium supplemented
with glucose (20 g L–1), yeast extract (6 g L–1) and
kanamycin (50 mg L–1) in a tri-baffled flask (250-mL). For
culturing cells for small scale biotransformation, the cells
were grown for about 2 h at 37 °C and 250 rpm to reach an
OD600 of 0.6-0.8, and then IPTG (0.5 mM final
concentration) was added to induce the expression of
enzymes. The cells were further grown overnight (12-13 h)
at 22 °C to reach late exponential phase, and they were
harvested by centrifugation (3500 g, 10 min). For culturing
cells for preparative biotransformation, the cells in 50 mL
M9 medium were grown for 7-9 h at 37 °C and then
transferred into 2 × 500 mL M9 medium (containing
glucose, yeast extract and kanamycin) in two baffled flasks
(2.5-L, TunairTM). The cells were grown for about 1-2 h at
37 °C and 250 rpm to reach an OD600 of 0.6-0.8, and then
IPTG (0.5 mM) was added. The cells were further grown
overnight (13-15 h) at 22 °C to reach late exponential
phase, and they were harvested by centrifugation (3500 g,
20 min). The cell pellets were resuspended in KP buffer to
measure optical density at 600 nm and used as resting cells
for biotransformation.
Analytical Methods
HPLC analysis of 4a-4l was performed on a Shimadzu
prominence HPLC system with a photodiode array detector
and a reverse-phase Agilent Poroshell 120 SB-C18 column
(150 × 4.6 mm, 2.7 µm) at 25 °C. Mobile phase: 50%
water with 0.1% TFA: 50% acetonitrile. Flow rate: 0.5 ml
min–1. The concentration was determined by comparison of
peak areas at 210 nm to those on the calibration curve of
the authentic compound. Retention times: phenethylamine
(internal standard) 3.2 min, 4a 4.8 min, 4b 5.0 min, 4c 5.1
min, 4d 5.0 min, 4e 6.0 min, 4f 6.1 min, 4g 6.4 min, 4h 6.5
min, 4i 5.7 min, 4j 5.7 min, 4k 4.8 min, 4l 4.7 min.
GC-FID analysis of 1a-1l, 2a and 3a was performed on
an Agilent 7890A gas chromatograph system with an FID
detector. Column: Agilent HP-5 (30 m × 0.32 mm × 0.25
Enzyme Activity Test with Recombinant E. coli Cells
o
mm). Temperature program: start at 70 C, increase to 200
Specific activity for epoxidation of 1a: Freshly prepared E.
coli (StyABC-EcALDH) cells were resuspended to 2 g
cdw L–1 in KP buffer (200 mM, pH 8.0) containing glucose
(1%, w/v). 1 mL of the cell suspension was mixed with 1
mL n-hexadecane containing 1a (20 mM) in a flask and
incubated at 300 rpm and 30 °C for 30 min. Organic phase
sample (100 µl) was separated by centrifugation (13000 g,
3 min), diluted with 900 µl ethyl acetate, and analyzed by
GC-FID to quantify 1a. The specific activity in U (g cdw)–
1 is calculated based on the decrease of 1a.
Specific activity for isomerization of 2a: Freshly
prepared E. coli (StyABC-EcALDH) cells were
resuspended to 0.2 g cdw L–1 in KP buffer (200 mM, pH
8.0). 1 mL of the cell suspension was mixed with 1 mL n-
hexadecane containing 2a (100 mM) in a flask and
incubated at 300 rpm and 30 °C for 15 min. Organic phase
sample (100 µl) was separated by centrifugation (13000 g,
3 min), diluted with 900 µl ethyl acetate, and analyzed by
GC-FID to quantify 2a. The specific activity in kU (g
cdw)–1 is calculated based on the decrease of 2a.
Specific activity for oxidation of 3a: Freshly prepared E.
coli (StyABC-EcALDH) cells were resuspended to 2 g
cdw L–1 in KP buffer (200 mM, pH 8.0). 1 mL of the cell
suspension was mixed with 1 mL n-hexadecane containing
3a (20 mM) in a flask and incubated at 300 rpm and 30 °C
for 15 min. Aqueous phase sample (100 µl) was separated
by centrifugation (13000 g, 3 min), diluted with 400 µl
TFA solution (0.5%) and 500 µl acetonitrile, and analyzed
by HPLC to quantify 4a. The specific activity in U (g
cdw)–1 is calculated based on the formation of 4a.
o
o
o
oC at 25 C min–1, increase to 250 C at 50 C min–1, hold
for 1 min, and then increase to 270 C at 20 C min−1. The
concentration was determined by comparison of peak areas
to those on the calibration curve of the authentic compound.
Retention times: benzyl alcohol (internal standard) 2.8 min,
1a 2.2 min, 1b 2.2 min, 1c 2.2 min, 1d 2.2 min, 1e 3.1 min,
1f 3.1 min, 1g 3.5 min, 1h 3.5 min, 1i 2.6 min, 1j 2.6 min,
1k 3.4 min, 1l 3.4 min.
o
o
Chiral HPLC analysis of 4m-4p (concentration) was
performed on the same HPLC system with a reverse-phase
Daicel Chiralpak AD-3R column (150 × 4.6 mm, 3 µm) at
15 °C. The concentration was determined by comparison
of peak areas at 210 nm to those on the calibration curve of
authentic compound. Method A: mobile phase consisting
of 80% water with 0.1% TFA: 20% acetonitrile was
delivered at 1.0 ml min–1. Retention times: benzyl alcohol
(internal standard) 5.8 min, (R)-4m 13.9 min, (S)-4m 14.6
min, (R)-4n 19.2 min, (S)-4n 19.9 min, (R)-4p 32.3 min,
(S)-4p 33.4 min. Method B: mobile phase consisting of
70% water with 0.1% TFA: 30% acetonitrile was delivered
at 1.0 ml min–1. Retention times: benzyl alcohol (internal
standard) 3.8 min, (R)-4o 12.2 min, (S)-4o 13.0 min.
The ee values of (S)-4m-4p were measured with another
chiral HPLC analysis method using a Daicel Chiralpak
AD-H column (250 × 4.6 mm, 5 µm) at 25 °C. Mobile
phase consisting of 90% n-hexane with 0.1% TFA: 10%
isopropanol was delivered at 1.0 ml min–1. Retention
times: (R)-4m 5.7 min, (S)-4m 6.3 min, (R)-4n 5.7 min,
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201700416
Advanced Synthesis & Catalysis
General Procedure for Biotransformation on Small
Scale
3-Fluorophenylacetic acid 4c: white solid; 225 mg
(73% yield); H NMR (400 MHz, CDCl3): δ = 7.32–7.26
1
(m, 1H), 7.07–6.96 (m, 3H), 3.65 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 176.8, 162.8 (d, J = 244.8 Hz),
135.4 (d, J = 7.9 Hz), 130.1 (d, J = 8.2 Hz), 125.1 (d, J =
2.9 Hz), 116.4 (d, J = 21.8 Hz), 114.4 (d, J = 20.6 Hz),
40.6 ppm.
Freshly prepared E. coli (StyABC-EcALDH) cells were
resuspended to 10 g cdw L–1 in KP buffer (200-400 mM,
pH 6.0-9.0, 0-2% glucose). 2 mL of the cell suspension
was mixed with 0.2-2 mL of different organic phase
containing 1a (150 mM) in a flask and incubated at 300
rpm and 22-37 °C for 10 h. Aqueous phase samples (20 µl)
were separated by centrifugation (13000 g, 3 min), diluted
with 480 µl TFA solution (0.5%) and 500 µl acetonitrile
(with 2 mM phenethylamine), and analyzed by HPLC to
quantify 4a.
4-Fluorophenylacetic acid 4d: white solid; 234 mg
1
(76% yield); H NMR (400 MHz, CDCl3): δ = 7.26–7.23
(m, 2H), 7.04–7.00 (m, 2H), 3.63 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 177.4, 162.2 (d, J = 244.4 Hz),
130.9 (d, J = 8.0 Hz), 128.9 (d, J = 3.4 Hz), 115.5 (d, J =
21.3 Hz), 40.1 ppm.
Freshly prepared E. coli (StyABC-ADH9v1) cells were
resuspended to 15-20 g cdw L–1 in KP buffer (200 mM, pH
8.0, 0-1% glucose). 2 mL of the cell suspension was mixed
with 0.4 mL of n-hexadecane containing 1a (20 mM) in a
flask and incubated at 300 rpm and 30 °C for 24 h.
Aqueous phase samples (100 µl) were separated by
centrifugation (13000 g, 3 min), diluted with 400 µl TFA
solution (0.5%) and 500 µl acetonitrile (with 2 mM benzyl
alcohol), and analyzed by HPLC to quantify 4m.
3-Chlorophenylacetic acid 4e: white solid; 276 mg
1
(81% yield); H NMR (400 MHz, CDCl3): δ = 7.30–7.24
(m, 3H), 7.18–7.16 (m, 1H), 3.63 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 176.2, 135.0, 134.5, 129.9, 129.6,
127.6, 127.6, 40.4 ppm.
4-Chlorophenylacetic acid 4f: white solid; 256 mg
1
(75% yield); H NMR (400 MHz, CDCl3): δ = 7.32–7.29
(m, 2H), 7.23–7.20 (m, 2H), 3.62 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 176.8, 133.4, 131.6, 130.7, 128.8,
40.2 ppm.
General Procedure for Preparation of Arylacetic Acids
by Biotransformation of Styrene Derivatives with E.
coli (StyABC-EcALDH)
3-Bromophenylacetic acid 4g: white solid; 166 mg
1
(77% yield); H NMR (400 MHz, CDCl3): δ = 7.45–7.41
(m, 2H), 7.23–7.18 (m, 2H), 3.62 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 176.9, 135.3, 132.5, 130.6, 130.2,
128.1, 122.6, 40.5 ppm.
Freshly prepared E. coli (StyABC-EcALDH) cells were
resuspended to 15 g cdw L–1 in KP buffer (400 mM, pH
8.0, 0.5% glucose). 40-100 mL of the cell suspension was
mixed with 8-20 mL of EO in a tri-baffled flask (250-500
mL). 1a (10 mmol), 1b-1f (2 mmol), 1g-1h (1 mmol), or
1i-1l (2 mmol) was added to start the reaction at 300 rpm
and 30 °C for 10 h. Aqueous phase samples (50 µl) were
separated by centrifugation (13000 g, 3 min), diluted with
450 µl TFA solution (0.5%) and 500 µl acetonitrile (with 2
mM phenethylamine), and analyzed by HPLC to quantify
4a-4l. At the end of the reaction, the mixture was subjected
to centrifugation (4000 g, 15 min) to collect the aqueous
phase. The reaction flask, the cells and EO were washed
with water (10-20 mL). The aqueous phase and washed
water was combined, adjusted to pH ≤ 2 with HCl,
saturated with NaCl, and then extracted with ethyl acetate
(3 × 50-100 mL). The ethyl acetate was collected, dried
over Na2SO4, and subjected to evaporation by using a
rotary evaporator. The crude product was purified by flash
chromatography on a silica gel column with an eluent
consisting of n-hexane: ethyl acetate of 3-5: 1 and acetic
acid (0.5% as additive) (Rf = 0.2-0.3). The collected
fractions were subjected to GC-FID analysis to confirm the
purity. The desired fractions were combined, subjected to
evaporation, and dried overnight under vacuum.
4-Bromophenylacetic acid 4h: white solid; 153 mg
1
(71% yield); H NMR (400 MHz, CDCl3): δ = 7.48–7.44
(m, 2H), 7.16–7.14 (m, 2H), 3.61 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 177.4, 132.1, 131.8, 131.1, 121.5,
40.4 ppm.
3-Methylphenylacetic acid 4i: white solid; 201 mg
1
(67% yield); H NMR (400 MHz, CDCl3): δ = 7.26–7.21
(m, 1H), 7.10–7.08 (m, 3H), 3.61 (s, 2H), 2.35 (s, 3H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 177.7, 138.4,
133.2, 130.1, 128.6, 128.1, 126.4, 41.0, 21.3 ppm.
4-Methylphenylacetic acid 4j: white solid; 168 mg
1
(56% yield); H NMR (400 MHz, CDCl3): δ = 7.19–7.13
(m, 4H), 3.61 (s, 2H), 2.34 (s, 3H) ppm; 13C NMR (100
MHz, CDCl3): δ = 178.0, 137.0, 130.2, 129.4, 129.2, 40.6,
21.1 ppm.
3-Methoxyphenylacetic acid 4k: white solid; 219 mg
1
(66% yield); H NMR (400 MHz, CDCl3): δ = 7.27–7.23
(m, 1H), 6.88–6.82 (m, 3H), 3.80 (s, 3H), 3.63 (s, 2H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 177.5, 159.8,
134.7, 129.6, 121.7, 115.1, 112.9, 55.2, 41.0 ppm.
4-Methoxyphenylacetic acid 4l: white solid; 173 mg
1
(52% yield); H NMR (400 MHz, CDCl3): δ = 7.21–7.19
(m, 2H), 6.88–6.86 (m, 2H), 3.80 (s, 3H), 3.59 (s, 2H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 178.0, 158.9,
130.4, 125.3, 114.1, 55.3, 40.1 ppm.
Phenylacetic acid 4a: white solid; 1116 mg (82%
1
yield); H NMR (400 MHz, CDCl3): δ = 7.31–7.05 (m,
5H), 3.66 (s, 2H) ppm; 13C NMR (100 MHz, CDCl3): δ =
177.9, 133.3, 129.4, 128.7, 127.4, 41.1 ppm.
General Procedure for Preparation of (S)-2-
Arylpropionic Acids by Biotransformation of 2-
Arylpropenes with E. coli (StyABC-ADH9v1)
2-Fluorophenylacetic acid 4b: white solid; 219 mg
1
(71% yield); H NMR (400 MHz, CDCl3): δ = 7.31–7.22
(m, 2H), 7.13–7.05 (m, 2H), 3.72 (s, 2H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 176.5, 161.1 (d, J = 245.5 Hz),
131.5 (d, J = 3.8 Hz), 129.4 (d, J = 8.1 Hz), 124.2 (d, J =
3.7 Hz), 120.7 (d, J = 15.6 Hz), 115.4 (d, J = 21.4 Hz),
34.2 (d, J = 3.1 Hz) ppm.
Freshly prepared E. coli (StyABC-ADH9v1) cells were
resuspended to 20 g cdw L–1 in KP buffer (200 mM, pH
8.0). 100 mL of the cell suspension was mixed with 10 mL
of n-hexadecane in a tri-baffled flask (500 mL). 1m (2
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201700416
Advanced Synthesis & Catalysis
mmol), or 1n-1p (0.5 mmol) was added to start the
reaction at 300 rpm and 30 °C for 24 h. Aqueous phase
samples (100 µl) were separated by centrifugation (13000
g, 3 min), diluted with 400 µl TFA solution (0.5%) and
500 µl acetonitrile (with 2 mM benzyl alcohol), and
analyzed by chiral HPLC to quantify the concentration and
ee of 4m-4p. At the end of the reaction, the mixture was
subjected to centrifugation (4000 g, 15 min) to collect the
aqueous phase. The reaction flask, the cells and n-
hexadecane were washed with water (20 mL). The aqueous
phase and washed water was combined, adjusted to pH ≤ 2
with HCl, saturated with NaCl, and then extracted with
ethyl acetate (3 × 100 mL). The ethyl acetate was collected,
dried over Na2SO4, and subjected to evaporation by using a
rotary evaporator. The crude product was purified by flash
chromatography on a silica gel column with an eluent
consisting of n-hexane: ethyl acetate of 5: 1 and acetic acid
(0.5% as additive) (Rf = 0.2-0.3). The collected fractions
were subjected to GC-FID analysis to confirm the purity.
The desired fractions were combined, subjected to
evaporation (n-heptane was added to remove acetic acid by
forming azeotrope), and dried overnight under vacuum.
(S)-2-Phenylpropanoic acid (S)-4m: colorless oil; 195
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20
mg (65% yield); 98% ee; [α]D = +70 (c = 0.5, CHCl3)
{lit.[23] [α]D25 = +66.1 (c = 1.8, CHCl3), 96% ee}; 1H NMR
(400 MHz, CDCl3): δ = 7.37–7.26 (m, 5H), 3.78–3.73 (q, J
= 7.2 Hz, 1H), 1.54–1.52 (d, J = 7.2 Hz, 3H) ppm; 13C
NMR (100 MHz, CDCl3): δ = 180.9, 139.8, 128.7, 127.6,
127.4, 45.4, 18.1 ppm.
(S)-4-Fluoro-α-methylphenylacetic acid (S)-4n: white
20
solid; 41 mg (49% yield); 97% ee; [α]D = +62 (c = 0.5,
18
CHCl3) {lit.[23] [α]D = +53.5 (c = 0.62, CHCl3), 98.4%
ee}; 1H NMR (400 MHz, CDCl3): δ = 7.30–7.26 (m, 2H), δ
= 7.04–6.99 (m, 2H), 3.75–3.70 (q, J = 7.2 Hz, 1H), 1.51–
1.49 (d, J = 7.2 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3): δ = 180.5, 162.1 (d, J = 244.5 Hz), 135.4 (d, J =
3.2 Hz), 129.1 (d, J = 8.0 Hz), 115.5 (d, J = 21.2 Hz), 44.6,
18.2 ppm.
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(S)-4-Chloro-α-methylphenylacetic acid (S)-4o: white
20
solid; 42 mg (46% yield); 92% ee; [α]D = +59 (c = 0.5,
CHCl3) {lit.[23] [α]D18 = +66.3 (c = 0.9, CHCl3), 98.5% ee};
1H NMR (400 MHz, CDCl3): δ = 7.31–7.24 (m, 4H), 3.74–
3.69 (q, J = 7.2 Hz, 1H), 1.51–1.49 (d, J = 7.2 Hz, 3H)
ppm; 13C NMR (100 MHz, CDCl3): δ = 180.2, 138.1,
133.3, 129.0, 128.8, 44.8, 18.1 ppm.
(S)-α,4-Dimethylphenylacetic acid (S)-4p: white solid;
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43 mg (52% yield); 98% ee; [α]D20 = +69 (c = 0.5, CHCl3)
14
1
{lit.[23] [α]D = +66.4 (c = 0.711, CHCl3), 99% ee}; H
NMR (400 MHz, CDCl3): δ = 7.26–7.14 (m, 4H), 3.76–
3.70 (q, J = 7.2 Hz, 1H), 2.34 (s, 3H), 1.52–1.50 (d, J = 7.2
Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ = 180.8,
137.1, 136.8, 129.4, 127.5, 45.0, 21.0, 18.1 ppm.
Acknowledgements
This work was supported by Ministry of Education of Singapore
through an AcRF Tier 1 Grant (Project No. R-279-000-477-112)
and National University of Singapore through Synthetic Biology
for Clinical and Technological Innovation (SynCTI) Program.
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Mihovilovic, U. T. Bornscheuer, ChemCatChem 2013,
5, 3524–3528; c) S. Schmidt, C. Scherkus, J. Muschiol,
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This article is protected by copyright. All rights reserved.

10.1002/adsc.201700416
Advanced Synthesis & Catalysis
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10.1002/adsc.201700416
Advanced Synthesis & Catalysis
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Regio- and Stereoselective Oxidation of Styrene
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