Enantioselective trans-dihydroxylation of aryl olefins by cascade biocatalysis with recombinant escherichia coli coexpressing monooxygenase and epoxide hydrolase

Article abstract of DOI:10.1021/cs400992z

Cascade biocatalysis via intracellular epoxidation and hydrolysis was developed as a green and efficient method for enantioselective dihydroxylation of aryl olefins to prepare chiral vicinal diols in high ee and high yield. Escherichia coli (SSP1) coexpressing styrene monooxygenase (SMO) and epoxide hydrolase SpEH was developed as a simple and efficient biocatalyst for S-enantioselective dihydroxylation of terminal aryl olefins 1a-15a to give (S)-vicinal diols 1c-15c in high ee (97.5-98.6% for 10 diols; 92.2-93.9% for 3 diols) and high yield (91-99% for 6 diols; 86-88% for 2 diols; 67% for 3 diols). Combining SMO and epoxide hydrolase StEH showing complementary regioselectivity to SpEH as a biocatalyst for the cascade biocatalysis gave rise to R-enantioselective dihydroxylation of aryl olefins, being the first example of this kind of reversing the overall enantioselectivity of cascade biocatalysis. E. coli (SST1) coexpressing SMO and StEH was also engineered as a green and efficient biocatalyst for R-dihydroxylation of terminal aryl olefins 1a-15a to give (R)-vicinal diols 1c-15c in high ee (94.2-98.2% for 7 diols; 84.2-89.9% for 6 diols) and high yield (90-99% for 6 diols; 85-89% for 5 diols; 65% for 1 diol). E. coli (SSP1) and E. coli (SST1) catalyzed the trans-dihydroxylation of trans-aryl olefin 16a and cis-aryl olefin 17a with excellent and complementary stereoselectivity, giving each of the four stereoisomers of 1-phenyl-1,2- propanediol 16c in high ee and de, respectively. Both strains catalyzed the trans-dihydroxylation of aryl cyclic olefins 18a and 19a to afford the same trans-cyclic diols (1R,2R)-18c and (1R,2R)-19c, respectively, in excellent ee and de. This type of cascade biocatalysis provides a tool that is complementary to Sharpless dihydroxylation, accepting cis-alkene and offering enantioselective trans-dihydroxylation.

Full text of DOI:10.1021/cs400992z

Research Article
pubs.acs.org/acscatalysis
Enantioselective trans-Dihydroxylation of Aryl Olefins by Cascade
Biocatalysis with Recombinant Escherichia coli Coexpressing
Monooxygenase and Epoxide Hydrolase
Shuke Wu,†,‡ Yongzheng Chen,† Yi Xu,† Aitao Li,† Qisong Xu,† Anton Glieder,§ and Zhi Li*,†,‡
†Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585
‡Singapore-MIT Alliance, National University of Singapore, 4 Engineering Drive 3, Singapore 117583
§Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria
S
* Supporting Information
ABSTRACT: Cascade biocatalysis via intracellular epoxidation
and hydrolysis was developed as a green and efficient method for
enantioselective dihydroxylation of aryl olefins to prepare chiral
vicinal diols in high ee and high yield. Escherichia coli (SSP1)
coexpressing styrene monooxygenase (SMO) and epoxide
hydrolase SpEH was developed as a simple and efficient
biocatalyst for S-enantioselective dihydroxylation of terminal
aryl olefins 1a−15a to give (S)-vicinal diols 1c−15c in high ee
(97.5−98.6% for 10 diols; 92.2−93.9% for 3 diols) and high yield
(91−99% for 6 diols; 86−88% for 2 diols; 67% for 3 diols).
Combining SMO and epoxide hydrolase StEH showing
complementary regioselectivity to SpEH as a biocatalyst for the
cascade biocatalysis gave rise to R-enantioselective dihydrox-
ylation of aryl olefins, being the first example of this kind of reversing the overall enantioselectivity of cascade biocatalysis. E. coli
(SST1) coexpressing SMO and StEH was also engineered as a green and efficient biocatalyst for R-dihydroxylation of terminal
aryl olefins 1a−15a to give (R)-vicinal diols 1c−15c in high ee (94.2−98.2% for 7 diols; 84.2−89.9% for 6 diols) and high yield
(90−99% for 6 diols; 85−89% for 5 diols; 65% for 1 diol). E. coli (SSP1) and E. coli (SST1) catalyzed the trans-dihydroxylation
of trans-aryl olefin 16a and cis-aryl olefin 17a with excellent and complementary stereoselectivity, giving each of the four
stereoisomers of 1-phenyl-1,2-propanediol 16c in high ee and de, respectively. Both strains catalyzed the trans-dihydroxylation of
aryl cyclic olefins 18a and 19a to afford the same trans-cyclic diols (1R,2R)-18c and (1R,2R)-19c, respectively, in excellent ee and
de. This type of cascade biocatalysis provides a tool that is complementary to Sharpless dihydroxylation, accepting cis-alkene and
offering enantioselective trans-dihydroxylation.
KEYWORDS: biocatalysis, biotransformations, cascade catalysis, dihydroxylation, enantioselectivity, epoxide hydrolase, monooxygenase,
vicinal diol
enantioselective cascade biocatalysis have been reported.11−27
Nevertheless, it is still very important to develop a new type of
INTRODUCTION
■
One-pot cascade catalysis is an important tool for sustainable
enantioselective cascade biocatalysis for practical synthesis.
chemical manufacturing, since it could avoid the energy- and
We recently developed a novel type of enantioselective
time-consuming, yield-decreasing, waste-producing, and labor-
cascade biocatalysis for trans-dihydroxylation of olefins to
demanding separation and purification of the intermediates in
traditional multistep synthesis.1−5 Compared with cascade
prepare enantiopure vicinal diols via one-pot cascade
epoxidation and hydrolysis, a formal dihydroxylation that is
chemical catalysis, cascade biocatalysis has several distinctive
features: it can be easily combined in one pot because of the
similar reaction conditions of enzyme catalysis; it is nontoxic;
different from dioxygenase-catalyzed dihydroxylation. Cells of
Sphingomonas sp. HXN-200 containing a P450 monooxyge-
nase28−31 and epoxide hydrolase (EH)32−34 were used to
catalyze the enantioselective trans-dihydroxylation of several
cyclic olefins.35 A tandem biocatalysts system consisting of the
Escherichia coli cells expressing styrene monooxygenase
and it is often highly chemo-, regio-, and enantioselective.6−10
Recently, enantioselective cascade biocatalysis has received
increasing attention for the synthesis of enantiopure com-
pounds for chiral pharmaceutical and fine chemical manufactur-
ing. Although cascade biocatalysis exists naturally in the
metabolic pathways in cells, it is necessary to develop
nonnatural cascade biocatalysis for the synthesis of nonnatural
chemical compounds. Over the years, several types of
Received: October 29, 2013
Revised: December 4, 2013
© XXXX American Chemical Society
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Research Article
Scheme 1. Enantioselective Dihydroxylation of Aryl Olefins 1a−15a with E. coli (SSP1) Coexpressing Styrene Monooxygenase
(SMO) and Epoxide Hydrolase from Sphingomonas sp. HXN-200 (SpEH) To Produce (S)-Vicinal Diols (S)-1c−15c and with E.
coli (SST1) Coexpressing SMO and Epoxide Hydrolase from Solanum tuberosum (StEH) To Produce (R)-Vicinal Diols (R)-1c−
15c, Respectively
(SMO)36−42 and the cell-free extract containing the EH
(SpEH)32−34 of Sphingomonas sp. HXN-200 was engineered
for one-pot simultaneous S-enantioselective dihydroxylation of
aryl olefins;43 lipase-mediated epoxidation, and EH-catalyzed
epoxide hydrolysis were combined for the enantioselective
trans-dihydroxylation of cyclic olefins via one-pot sequential
epoxidation and hydrolysis.44 Although asymmetric cis-
dihydroxylation of olefins can be achieved in one step by
Sharpless dihydroxylation using a heavy metal oxide catalyst
with chiral ligands,45 chemical trans-dihydroxylation of olefins
requires two reaction steps: epoxidation of olefins46 and
subsequent hydrolysis of epoxides,47,48 which needs the
separation of toxic and unstable epoxide intermediates and
also utilizes toxic metals (e.g., osmium, cobalt). Thus, one-pot
cascade biocatalysis for trans-dihydroxylation of alkenes provide
a greener and more efficient synthetic method for the
preparation of enantiopure vicinal diols than the corresponding
two-step chemical catalysis. It is also a complementary tool to
Sharpless dihydroxylation.
We are interested in further developing this type of cascade
biocatalysis as a practical method for the preparation of
enantiopure vicinal diols that are useful and valuable synthetic
intermediates for many pharmaceuticals, bioactive compounds,
and chiral reagents.49−58 A group of enantiopure vicinal diols
are selected as target compounds (Scheme 1−4). For example,
(S)-1-phenyl-1,2-ethanediol, 1c, is a crucial synthetic precursor
for pharmaceutical (R)-fluoxetine,50 chiral phosphoramidite
ligand,51 and an auxiliary for stereoselective glycosylation;52
(R)-1-(4-fluorophenyl)-1,2-ethanediol, 2c, is an intermediate
for preparing the cholesterol-lowering medicine Ezetimibe;53
(R)-1-(3-chlorophenyl)-1,2-ethanediol, 9c, is a key chiral
synthon for β3-adrenergic agonists;54 (S)-1-(3-methoxyphen-
yl)-1,2-ethanediol, 12c, is a chiral ligand for chromium complex
catalysts;55 (1R,2S)-phenylpropanediol, 16c, and (1S,2S)-16c
are useful for the synthesis of muscle relaxant phenyl-
carbanate56 and selegiline,57 respectively; and (1R,2R)-
indanediol, 18c, can be easily converted to (1S)-amino-(2R)-
indanol for the synthesis of the anti-HIV drug Indinavir.58 Thus
far, the reported cascade biocatalysis system for the
dihydroxylation of olefins is relatively complicated, and its
efficiency needs to be further improved for practical application,
the substrate scope is not fully explored, and the dihydrox-
ylation is only S-selective.
Here, we report the development of E. coli (SSP1) cells
coexpressing SMO and SpEH as a simpler and more efficient
biocatalyst for S-enantioselective dihydroxylation of 15 terminal
aryl olefins, 1a−15a, to produce the corresponding (S)-vicinal
diols 1c−15c in high ee and good yield. We also report the
development of E. coli (SST1) cells coexpressing SMO and
another EH (StEH) with the complementary regioselectivity of
SpEH as a simple and efficient biocatalyst for R-enantioselective
dihydroxylation of 15 terminal aryl olefins, 1a−15a, to produce
the corresponding (R)-vicinal diols 1c−15c in high ee and
good yield. E. coli (SSP1) and E. coli (SST1) cells are further
explored for the highly enantioselective trans-dihydroxylation of
nonterminal aryl olefins 16a−17a to synthesize all four
enantiomers of 1-phenyl-1,2-propanediol 16c and of aryl cyclic
olefins 18a−19a to prepare (1R,2R)-trans-diols 18c−19c.
RESULTS AND DISCUSSION
■
Genetic Engineering of E. coli Coexpressing SMO and
SpEH for S-Selective Dihydroxylation of Styrenes. SMO
was chosen as the enzyme for epoxidation of olefins in the first
step of the cascade because it is a well-known enzyme for the
epoxidation of styrenes to give (S)-epoxides in high ee.36−42 In
addition, the epoxidation of styrenes with recombinant E. coli
expressing SMO has been developed as one of the most
productive biocatalytic oxyfunctionalization processes.41,42 EH
from Sphingomonas sp. HXN-200 (SpEH) was used as the
enzyme for the hydrolysis of the epoxides in the second step of
the cascade because this EH is known to hydrolyze (S)-styrene
oxides at the “β” position to give (S)-diols in high ee with the
retention of configuration.32−34 The E. coli strain expressing
SpEH was also developed as an efficient catalyst for the
preparation of enantiopure epoxides by kinetic resolution.34
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Figure 1. (a) Genetic constructions of and dihydroxylation with three different E. coli strains coexpressing SMO (StyA and StyB) and SpEH; (b)
genetic constructions of and dihydroxylation with three different E. coli strains coexpressing SMO (StyA and StyB) and StEH. The dihydroxylation of
100 mM styrene, 1a, to diol, 1c, was performed with resting cells (10 g cdw/L) of the corresponding E. coli strain at 30 °C in a two-liquid-phase
system (n-hexadecane/KP buffer = 1:1).
To develop recombinant E. coli containing both SMO and
SpEH as a simple and efficient catalyst system for the
dihydroxylation of olefins, the two coding sequences of SMO
(styA and styB) were amplified from pSPZ10 plasmid,37 and the
gene of SpEH was amplified from the genome of the
Sphingomonas sp. HXN-200.34 To optimize the expression
level of the three genes, we engineered three different
expression cassettes on the commercially available plasmid
pRSFduet (Figure 1a): SSP1, an artificial operon of styA, styB,
and spEH controlled by one T7 promoter; and SSP2-1 and
SSP2-2, where styA, styB, and spEH are under the control of
two T7 promoters. These plasmids were transformed into E.
coli T7 expression cells to give E. coli (SSP1), E. coli (SSP2-1),
and E. coli (SSP2-2), respectively. These strains were grown in
TB medium, and IPTG was added to induce protein
expression. As a result, all three strains were able to coexpress
SMO (StyA and StyB) and SpEH, but at different levels. The
strains were examined for biotransformation of 100 mM
styrene, 1a, in a two-liquid-phase system consisting of
potassium phosphate (KP) buffer and n-hexadecane (1:1). E.
coli (SSP1) showed the best results, producing 65 mM (S)-1-
phenylethane-1,2-diol, 1c, in 99% ee at 5 h (Figure 1a). The
other two strains also gave (S)-1c, but in lower concentrations.
In comparison with other two strains, E. coli (SSP1) has a
higher ratio of SMO/SpEH, which is desirable for the cascade
dihydroxylation because the catalytic efficiency of SpEH (kcat/
Km = 6.0 mM−1 s−1)34 is higher than that of SMO (kcat/Km =
4.2 mM−1 s−1).59 The superiority of E. coli (SSP1) is also
probably due to the more homogeneous expression of several
genes in one operon.60,61 E. coli (SSP1) was selected for further
development.
expressing StEH (5 g cdw/L) for the dihydroxylation of 100
mM styrene, 1a, in the same two-liquid-phase system as
described above. (R)-1-Phenyl-1,2-ethanediol, 1c, was formed
in 95% ee and 74% yield. To avoid the difficulty in cultivation
of yeast cells and the preparation of cells of two micro-
organisms, we started to engineer an E. coli strain coexpressing
SMO and StEH as a simple and efficient catalyst for R-selective
dihydroxylation of styrenes. E. coli (pSPZ10_pMS470Δ8) was
constructed by electronically transforming pMS470Δ8 (con-
taining StEH) to competent cells of E. coli (pSPZ10)
(containing styA and styB). During cell growth, SMO was
induced by adding 0.1% (v/v) DCPK, and StEH was induced
by adding 0.05 mM IPTG. The harvested cells were used at 20
g cdw/L for the dihydroxylation of 100 mM styrene 1a in the
two-liquid-phase system for 8 h to produce (R)-1-phenyl-1,2-
ethanediol, 1c, in 93% ee and 90% yield. The specific activity
was ∼25 U/g cdw, which is lower than that of E. coli (SSP1)
(50 U/g cdw).
To engineer a more active E. coli strain coexpressing SMO
and StEH for R-selective dihydroxylation of styrenes, the gene
of StEH was synthesized according to the reported potato
cDNA sequence65 with codon optimization for the expression
in E. coli. Similar to the engineering of SMO and SpEH, three
different expression cassettes of SMO and StEH were
constructed, and three strains E. coli (SST1), E. coli (SST2-
1), and E. coli (SST2-2) were obtained and evaluated for the
dihydroxylation (Figure 1b). E. coli (SST1) gave a specific
activity of 40 U/g cdw and produced 82 mM (R)-1-
phenylethane-1,2-diol, 1c, in 96% ee at 5 h in the
dihydroxylation of 100 mM styrene 1a at a cell density of 10
g cdw/L in the two-liquid-phase system. It is the best among
the three strains. It also showed higher activity than E. coli
(pSPZ10_pMS470Δ8). Thus, E. coli (SST1) was chosen for
further development as a powerful catalyst for R-enantiose-
lective dihydroxylation of styrenes, being complementary to E.
coli (SSP1) for the cascade biocatalysis.
Genetic Engineering of E. coli Coexpressing SMO and
StEH for R-Selective Dihydroxylation of Styrenes. To
engineer a cascade biocatalysis for R-selective dihydroxylation
of styrenes, the EH from Solanum tuberosum (StEH) was
selected as the enzyme for the hydrolysis step because StEH is
known to hydrolyze (S)-styrene oxides to offer (R)-diols by
opening at the α position (inversion of configuration).62−64
The regioselectivity for the hydrolysis with StEH is
complementary to that with SpEH. The concept was
successfully proven by using a tandem biocatalysts system
consisting of resting cells of E. coli (pSPZ10) expressing SMO
(5 g cdw/L) and Pichia pastoris CBS 7435_MutS_PotHis
Cell Growth and Dihydroxylation Activity of E. coli
(SSP1) and E. coli (SST1). E. coli (SSP1) strain was grown
easily in M9 medium, with glucose as the carbon source, in a
shaking flask, and SMO and SpEH were coexpressed by adding
IPTG as the inducer. The growth and activity of the cells were
monitored by taking samples at different time points for
measuring the optical density and dihydroxylation activity. As
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Figure 2. (a, b) Cell growth and specific activity for dihydroxylation of the recombinant strains: (a) E. coli (SSP1), (b) E. coli (SST1). Cells were
initially cultured at 37 °C, induced at 2 h by the addition of IPTG (0.5 mM), and then grown at 25 °C. The activities were based on 30 min
dihydroxylation of 10 mM styrene 1a with resting cells (1 g cdw/L) in aqueous buffer. (c) SDS−PAGE. Lane 1, protein marker; lane 2, cell-free
extract of E. coli (SST1) coexpressing SMO and StEH taken at 12 h; lane 3, cell-free extract of E. coli (SSP1) coexpressing SMO and SpEH taken at
12 h.
shown in Figure 2a, the cells reached a high density (5−6 g
cdw/L; cdw: cell dry weight) at 13−16 h. High specific
activities (70−80 U/g cdw) toward S-dihydroxylation of
styrene 1a were achieved at the late exponential growing
phase (11−13 h). In the SDS−PAGE of the cell free extract of
the E. coli (SSP1) taken at 12 h (Figure 2c, lane 3), StyA, StyB,
and SpEH are clearly visible.
E. coli (SST1) showed a similar cell growth curve and
reached high cell density (5−6 g cdw/L) at 13−16 h (Figure
2b). Good specific activities (40−50 U/g cdw) for R-
dihydroxylation of styrene 1a were also achieved at the late
exponential growing phase (12−14 h). The SDS−PAGE of the
cell free extract of the E. coli (SST1) taken at 12 h (Figure 2c,
lane 2) also clearly demonstrated the expressing of StyA, StyB,
and StEH in E. coli (SST1).
Asymmetric Dihydroxylation of Terminal Aryl Olefins
1a−15a to (S)-Vincinal Diols 1c−15c with Resting Cells
of E. coli (SSP1). To explore the synthetic potential of E. coli
(SSP1), the resting cells of the strain were employed for the
dihydroxylation of 20 mM styrene 1a and substituted styrenes
2a−15a (Scheme 1) in a two-liquid-phase system containing
KP buffer and n-hexadecane (1:1). The n-hexadecane phase acts
as a reservoir of the substrate and possible epoxide intermediate
to reduce their inhibition effect on the enzymes. As listed in
Table 1, (S)-vincinal diols 1c−15c were produced in high ee
from 1a−15a by the one-pot cascade epoxidation and
hydrolysis with resting cells of E. coli (SSP1). Importantly,
many vicinal diols, such as (S)-1c−4c, (S)-7c−10c, (S)-12c,
and (S)-13c, were produced in excellent ee (≥97.5%). Other
vicinal diols, including (S)-5c, (S)-11c, and (S)-14c, were
obtained in high ee (92.2−93.9%). The configurations of 1c−
15c were established by comparing bioproducts with the
standard diols that were either commercially available or
prepared via Sharpless asymmetric dihydroxylation (Figures
S2−S16, Supporting Information). The high S-enantioselectiv-
ity of dihydroxylation is due to the high S-enantioselectivity of
SMO-catalyzed epoxidation of styrenes and the high
regioselectivity of SpEH-catalyzed hydrolysis of (S)-epoxides
at the β position. Only two diols, (S)-6c and (S)-15c, were
produced in moderate ee (83.2 and 65.7%), possibly because of
the autohydrolysis of unstable epoxide 6b or the reduced
regioselectivity of SpEH in the hydrolysis of epoxide 15b.
Most of the dihydroxylations gave high conversion and high
yield. (S)-Vicinal diols 1c, 8c, 9c, 11c, 12c, and 13c were
obtained in 91−>99% yields; (S)-2c and (S)-5c were formed in
86−88% yields; and (S)-3c, (S)-6c, and (S)-10c were produced
in 67% yields. This confirmed that SMO and SpEH
coexpressed in the E. coli cells are very active for the cascade
biocatalysis. The specific activity for these S-dihydroxylations is
11−55 U/g cdw, with an exception for the S-dihydroxylation of
10a (8 U/g cdw). In the previously reported tandem
biocatalysts system, (S)-epoxide 1b accumulated as the
intermediate in the early stage of the biotransformation.43
Because of the high activity of SpEH in E. coli (SSP1), there
was no epoxide accumulated during the cascade biocatalysis
with resting cells of E. coli (SSP1). The time curve of the
dihydroxylation of 9a was a representative example (Figure S1,
Supporting Information), which clearly evidences this point. In
comparison with the previously reported tandem biocatalysts
(20 mM 1a was converted in 21 h with 2.5 g cdw/L of E. coli
cells expressing SMO and 20 g of protein/L of cell free extract
containing SpEH),43 the use of resting cells of E. coli (SSP1)
coexpressing SMO and SpEH provides a much simpler catalyst
and much higher catalytic efficiency: 20 mM 1a was converted
to (S)-1c in only 2 h with 10 g cdw/L of E. coli (SSP1) cells.
The S-dihydroxylations of 4a, 7a, 14a, and 15a with resting
cells of E. coli (SSP1) gave lower yields (25−34%). This is
mainly due to the low epoxidation activity of SMO toward
these substrates, which have either a strong electron-with-
drawing group or an ortho substitution.38−40
Asymmetric Dihydroxylation of Terminal Aryl Olefins
1a−15a to (R)-Vicinal Diols 1c−15c with Resting Cells of
E. coli (SST1). To explore the synthetic potential of another
strain, E. coli (SST1), the resting cells of the strain were used
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Table 1. Enantioselective Dihydroxylation of Aryl Olefins 1a−15a with Resting Cells of E. coli (SSP1) and E. coli (SST1),
Respectively
a
The reactions were performed with substrates 1a−15a (20 mM in organic phase) and resting cells (10 g cdw/L) in a two-liquid-phase system
b
consisting of KP buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1:1) at 30 °C for 8 h. Activity is the specific activity determined for the
initial 30 min. Conversion is the consumption of starting substrate, determined by normal phase HPLC analysis of the remaining substrate in the n-
hexadecane phase. Error limit: 3% of the state values. Yield is the analytical yield of the formation of diol product, determined by reversed phase
HPLC analysis of the product in the aqueous phase. Error limit: 3% of the state values. The ee value was determined by chiral HPLC analysis. Error
c
d
e
f
limit: 0.2% of the state values. Yield is slightly higher than conversion due to the error limit in the measurement of yield and conversion.
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for the R-dihydroxylation of 20 mM olefins 1a−15a (Scheme
1) in the same two-liquid-phase system as described above. The
results are listed in Table 1, and (R)-vicinal diols (R)-1c−15c
were produced from 1a−15a by the one-pot cascade
epoxidation and hydrolysis with resting cells of E. coli
(SST1). Many (R)-vicinal diols, such as (R)-1c−4c, (R)-8c,
(R)-9c, and (R)-11c, were produced in excellent ee (94.2−
98.2%). The diols (R)-5c−7c, (R)-10c, (R)-12c, and (R)-15c
were also formed in high ee (84.2−89.9%). The high R-
enantioselectivity of dihydroxylation is the combined result of
high S-enantioselectivity of SMO-catalyzed epoxidation of
styrenes and the high regioselectivity of StEH-catalyzed
hydrolysis of (S)-epoxides at the α position. Only (R)-13c
and (R)-14c were obtained in low ee (68.1 and 36.9%). The
low enantioselectivity was probably caused by the hindrance of
the ortho-substitution in epoxides 13b−14b for the hydrolysis
at the α position with StEH.
Scheme 2. Enantioselective Trans-Dihydroxylation of
Nonterminal Aryl Olefins 16a and 17a with E. coli (SSP1)
(expressing SMO and SpEH) To Produce Vicinal Diols
(1S,2R)-16c and (1S,2S)-16c, and with E. coli (SST1)
(expressing SMO and StEH) To Produce Vicinal Diols
(1R,2S)-16c and (1R,2R)-16c, Respectively
Most of the R-dihydroxylations with the resting cells of E. coli
(SST1) gave also high conversion and high yield. (R)-Vicinal
diols 1c, 2c, 8c, 9c, 11c, and 12c were obtained in 90−>99%
yields; (R)-3c, (R)-4c, (R)-5c, (R)-10c, and (R)-13c were
formed in 85−89% yields; and (R)-6c was produced in 65%
yield. This also confirmed that SMO and StEH coexpressed in
the E. coli cells are very active for the cascade biocatalysis. The
specific activity for these R-dihydroxylations is 15−43 U/g cdw,
with an exception for the R-dihydroxylations of 4a, 10a, and
11a (6−9 U/g cdw). Because of the high activity of StEH in E.
coli (SST1), no epoxide was accumulated in the dihydrox-
ylations of 1a−12a. Accumulation of expoxide was observed
only in the dihydroxylation of ortho-substituted olefins 13a−
15a. Here, the resting cells of E. coli (SST1) coexpressing SMO
and StEH were developed as a simple and efficient catalyst for
the synthesis of these (R)-vicinal diols via dihydroxylation,
being the first biocatalytic system for the R-enantioselective
dihydroxylations of styrenes. Clearly, E. coli (SSP1) and E. coli
(SST1) are excellent cascade biocatalysts with complementary
enantioselectivity for the dihydroxylation of styrene and its
derivatives. It is the first example to achieve the reversal of
overall enantioselectivity of cascade biocatalysis by changing the
regioselectivity in an individual reaction step. The concept can
be applied to solve the problem of lacking mirror-image
enzymes in biocatalysis, in addition to the discovery and
development of enantiocomplementary enzymes.66
hand, trans-dihydroxylation of β-methyl styrenes 16a and 17a
with E. coli (SSP1) afforded (1S,2R)-16c in 94.2% ee and 91.8%
de and (1S, 2S)-16c in 85.6% ee and >99% de, respectively.
The regioselectivity and the yield for the hydrolysis were
decreased, possibly as a result of the steric hindrance of a β-
methyl group in the epoxide intermediate to the hydrolysis at
the β position with SpEH. Nevertheless, the product ee and de
were still quite high. The great achievement here was the
production of all four stereoisomers of 1-phenyl-1,2-propane-
diol, 16c, in high ee and de by the trans-dihydroxylation of
trans-alkene 16a and cis-alkene 17a with the two comple-
mentary biocatalysts, respectively. In comparison, the elegant
Sharpless dihydroxylation has difficulty in transforming cis-
alkene, such as 17a, with high selectivity.45
Asymmetric Trans Dihydroxylation of Aryl Cyclic
Olefins 18a and 19a with Resting Cells of E. coli (SSP1)
and E. coli (SST1). The trans-dihydroxylation of cyclic aryl
olefins 18a and 19a (Scheme 3) was performed with resting
cells of E. coli (SSP1) and E. coli (SST1). As shown in Table 2,
E. coli (SSP1) gave (1R,2R)-18c and (1R,2R)-19c in very high
ee (>96%), de (>98%), and yield (73−80%) from 18a and 19a,
respectively. The configurations of 18c−19c were established
by comparing bioproducts with the standard diols that were
commercially available (Figures S19, S20, Supporting Informa-
tion). The unexpected change in the enantioselectivity of the
dihydroxylation was possibly caused by the change in the
regioselectivity of SpEH in E. coli (SSP1) to the cyclic epoxides
18b and 19b. On the other hand, E. coli (SST1) also produced
(1R,2R)-18c and (1R,2R)-19c in very high ee (>96%), de
(>98%), and yield (67−71%) from 18a and 19a, respectively.
These results demonstrated once again the unique potential of
E. coli (SSP1) and E. coli (SST1) in the asymmetric trans-
dihydroxylation of cis-alkenes. In comparison, Sharpless
dihydroxylation has difficulty with cis-alkenes as starting
materials and could not produce trans-vicinal diols (1R,2R)-
18c and (1R,2R)-19c from cis-alkenes 18a and 19a.45
The R-dihydroxylations of 7a, 14a, and 15a with resting cells
of E. coli (SST1) gave lower yields (10−19%). This is similar to
the cases with E. coli (SSP1) and due to the low activity of
SMO.
Asymmetric Trans Dihydroxylation of Nonterminal
Aryl Olefins 16a and 17a with Resting Cells of E. coli
(SSP1) and E. coli (SST1). To prepare vicinal diols with two
chiral centers and evidently distinguish our trans-dihydrox-
ylation with Sharpless cis-dihydroxylation, we tested the
dihydroxylation of nonterminal olefin substrates 16a and 17a
with our catalysts (Scheme 2). The trans-dihydroxylation was
also performed in a two-phase system with resting cells as
catalysts. As shown in Table 2, trans-dihydroxylation of 16a and
17a with E. coli (SST1) gave (1R,2S)-16c and (1R,2R)-16c in
excellent ee (>98%) and de (≥98%), respectively. The
configuration of 16c was established by comparing bioproducts
with the standard diols that were prepared via Sharpless
asymmetric dihydroxylation (Figures S17, S18, Supporting
Information). The yields (96 and 89%) and the specific
activities (15 and 20 U/g cdw) were also high. On the other
Asymmetric Dihydroxylation of Other Aryl Olefins
20a−22a with Resting Cells of E. coli (SSP1) and E. coli
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Table 2. Enantioselective Trans-Dihydroxylation of Nonterminal Aryl Olefins 16a, 17a and Aryl Cyclic Olefins 18a, 19a with
Resting Cells of E. coli (SSP1) and E. coli (SST1), Respectively
a
The reactions were performed with substrates 16a−19a (20 mM in organic phase) and resting cells (10 g cdw/L) in a two-liquid-phase system
b
consisting of KP buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1:1) at 30 °C for 8 h. Activity is the specific activity determined for initial
30 min. Conversion is the consumption of starting substrate, determined by normal phase HPLC analysis of the remaining substrate in the n-
hexadecane phase. Error limit: 3% of the state values. Yield is the analytical yield of the formation of diol product, determined by reversed phase
HPLC analysis of the product in the aqueous phase. Error limit: 3% of the state values. The ee value was determined by chiral HPLC analysis. Error
c
d
e
f
g
limit: 0.2% of the state values. The de value was determined by chiral HPLC analysis. Error limit: 0.2% of the state values. Error limit: 0.5% of the
state values.
Scheme 3. Enantioselective Trans-Dihydroxylation of Aryl
Cyclic Olefins 18a and 19a with E. coli (SSP1) (expressing
SMO and SpEH) or E. coli (SST1) (expressing SMO and
StEH) To Produce Vicinal Diols (1R,2R)-18c and (1R,2R)-
19c
(SSP1) afforded (S)-21c in 94.5% ee and (S)-22c in 97.6% ee,
respectively, but dihydroxylation of 21a and 22a with E. coli
(SST1) failed to produce (R)-21c and (R)-22c, which is
probably due to the poor α-regioselectivity of StEH to the
corresponding epoxide intermediates. The configurations of
20c−22c were established by comparing bioproducts with the
standard diols that were prepared via Sharpless asymmetric
dihydroxylation (Figure S21−23, Supporting Information).
Preparation of Vicinal Diols (S)-1c, (S)-2c, (S)-5c, (S)-9c,
(S)-12c, (R)-1c, (R)-2c, (R)-9c, (1R,2S)-16c, and (1R,2R)-16c
by Asymmetric Dihydroxylation of Aryl Olefins with
Resting Cells of E. coli (SSP1) or E. coli (SST1). To further
demonstrate the synthetic potential of trans-dihydroxylation via
cascade biocatalysis, we carried out the preparation of 10
valuable vicinal diols from 7 aryl olefins1a, 2a, 5a, 9a, 12a,
16a, 17aon a 50 mL scale with the resting cells of E. coli
(SSP1) or E. coli (SST1). The syntheses were performed at a
substrate concentration of 50 mM (based on total reaction
volume) in a modified two-phase system containing 45 mL of
aqueous KP buffer and 5 mL of n-hexadecane. The reactions
were monitored by TLC by checking the disappearance of the
substrates. After 5−8 h, the reactions were stopped, and the
products were separated and purified by flash chromatography.
All 10 useful and valuable vicinal diols(S)-1c, (S)-2c, (S)-5c,
(S)-9c, (S)-12c, (R)-1c, (R)-2c, (R)-9c, (1R,2S)-16c, and
(1R,2R)-16cwere obtained in high ee (92.4−98.6%), de (de
≥ 98%, if applicable), and good isolated yield (70.6−85.5%)
(Table 4). The dihydroxylation via cascade biocatalysis gave
around 0.3 g of product/g of cells. In the representative
procedure of Sharpless asymmetric dihydroxylation,45 1.4 g of
AD-mix-α was used for the dihydroxylation of 1 mmol of olefin,
corresponding to ∼0.1 g of product/g of catalyst. In our
cascade biocatalysis for trans-dihydroxylation of olefins, whole
cells are used as a less expensive and greener catalyst, molecular
oxygen is used as the less expensive and green oxidant, and
(SST1). We further tested E. coli (SSP1) and E. coli (SST1) for
the dihydroxylation of substituted olefins (20a−22a) (Scheme
4, Table 3) in a two-liquid-phase system with the resting cells as
catalysts. With two methyl groups on the β carbon, 20a could
not be converted to (S)-20c in high ee by E. coli (SSP1)
because of the huge steric hindrance at the β position for
hydrolysis with SpEH. On the other hand, dihydroxylation of
20a with E. coli (SST1) gave (R)-20c in 98.2% ee and 83%
yields. In contrast, dihydroxylation of 21a and 22a with E. coli
Scheme 4. Enantioselective Dihydroxylation of Aryl Olefins
20a−22a with E. coli (SSP1) (expressing SMO and SpEH) or
E. coli (SST1) (expressing SMO and StEH) To Produce
Vicinal Diols (R)-20c, (S)-21c, and (S)-22c
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Table 3. Enantioselective Dihydroxylation of Aryl Olefins 20a−22a with Resting Cells of E. coli (SSP1) and E. coli (SST1),
Respectively
a
The reactions were performed with substrates 20a−22a (20 mM in organic phase) and resting cells (10 g cdw/L) in a two-liquid-phase system
b
consisting of KP buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1:1) at 30 °C for 8 h. Activity is the specific activity determined for the
initial 30 min. Conversion is the consumption of starting substrate, determined by normal phase HPLC analysis of the remaining substrate in the n-
hexadecane phase. Error limit: 3% of the state values. Yield is the analytical yield of the formation of diol product, determined by reversed phase
HPLC analysis of the product in the aqueous phase. Error limit: 3% of the state values. The ee value was determined by chiral HPLC analysis. Error
c
d
e
f
limit: 0.2% of the state values. Yield is slightly higher than conversion because of the error limit in the measurement of yield and conversion.
Table 4. Preparation of (R)- or (S)-Vicinal Diols in High ee by Enantioselective Dihydroxylation of Aryl Alkenes with Resting
Cells of E. coli (SSP1) or E. coli (SST1)
isolated yield
a
b
c
sub
catalyst
time (h)
prod
(S)-1c
g
%
ee (%)
de (%)
prod./cells (g/g cdw)
d
1a
1a
2a
2a
5a
9a
9a
E. coli (SSP1)
E. coli (SST1)
E. coli (SSP1)
E. coli (SST1)
E. coli (SSP1)
E. coli (SSP1)
E. coli (SST1)
E. coli (SSP1)
E. coli (SST1)
E. coli (SST1)
5
5
6
5
8
8
8
6
7
8
0.295
0.289
0.299
0.325
0.279
0.326
0.304
0.358
0.313
0.300
85.5
83.8
76.7
80.7
73.4
75.6
70.6
85.3
82.3
78.8
96.3
95.8
96.7
96.7
92.4
96.5
96.3
96.8
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
98.2
0.30
0.29
0.30
0.33
0.28
0.33
0.30
0.36
0.31
0.30
(R)-1c
(S)-2c
(R)-2c
(S)-5c
(S)-9c
(R)-9c
12a
16a
17a
(S)-12c
(1R,2S)-16c
(1R,2R)-16c
e
>98
98.6
>99
a
The reactions were performed with substrates (50 mM based on total volume) and resting cells (20 g cdw/L) in a two-liquid-phase system (50 mL)
b
consisting of KP buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (9:1) at 30 °C. The ee value was determined by chiral HPLC analysis.
Error limit: 0.2% of the state values. The de value was determined by chiral HPLC analysis. Error limit: 0.2% of the state values. n.a.: not
applicable. Error limit: 0.5% of the state values.
c
d
e
water is used for the epoxide hydrolysis. The green and efficient
cascade biocatalysis provides practical syntheses of the useful
and valuable vicinal aryl diols in high ee and high yield.
Dihydroxylation of Styrene 1a with Growing Cells in
Fermentor. We further explored the potential of using
growing cells of the recombinant E. coli strain for the
dihydroxylation of aryl olefins. Dihydroxylation of styrene 1a
with E. coli (SST1) was chosen as a model reaction. To avoid
the possible environmental concerns and reduce the additional
cost, an organic phase (n-hexadecane) was not applied in the
growing cell experiment. Instead, styrene 1a was fed directly
and slowly into the reaction mixture to alleviate the toxicity of
styrene.
E. coli (SST1) was grown in a fermentor overnight to a cell
density of 7 g cdw/L, glucose was fed, and IPTG was added to
induce the enzyme expression. After 5 h of growth, the cell
density reached 20 g cdw/L, and styrene 1a was fed to start the
dihydroxylation. After 5 h of biotransformation with the
growing cells, 120 mM (16.6 g/L) (R)-1-phenyl-1,2-ethanediol
1c was produced in 96.2% ee with an average volumetric
productivity of 3.3 g/L/h for the reaction period. The
enantioselectivity of the dihydroxylation with growing cells
was the same as that with resting cells. Thus, the cascade
biocatalysis for enantioselective dihydroxylation can be
performed with either growing cells or resting cells as catalysts,
it can also be carried out in either aqueous phase or a two-phase
system, and it can be easily scaled up by using a fermentor. The
use of growing cells as the catalyst may further improve the
product titer and volumetric productivity. Further optimization
of the process could make the cascade biocatalysis even more
practical for the enantioselective trans-dihydroxylation of aryl
olefins.
CONCLUSION
■
Escherichia coli (SSP1) cells coexpressing styrene monoox-
ygenase (SMO) and epoxide hydrolase SpEH were developed
as a green and efficient biocatalyst for S-enantioselective
dihydroxylation of aryl olefins via intracellular cascade
epoxidation and hydrolysis. The S-enantioselectivity was
generated by SMO-catalyzed S-selective epoxidation and
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SpEH-catalyzed regioselective hydrolytic opening of the (S)-
epoxide at the β position. Dihydroxylation of terminal aryl
olefins 1a−15a with resting cells of E. coli (SSP1) offered (S)-
vicinal diols 1c−15c in high ee (97.5−98.6% for 10 diols;
92.2−93.9% for 3 diols) and high yield (91−99% for 6 diols;
86−88% for 2 diols; 67% for 3 diols).
Combining SMO and epoxide hydrolase StEH showing an α
opening of aryl epoxides as the catalyst for the cascade
biocatalysis gave rise to R-enantioselective dihydroxylation of
aryl olefins. E. coli (SST1) coexpressing SMO and StEH was
also engineered as a green and efficient biocatalyst for R-
dihydroxylation of aryl olefins, being complementary to E. coli
(SSP1). Dihydroxylation of terminal aryl olefins 1a−15a with
resting cells of E. coli (SST1) afforded (R)-vicinal diols 1c−15c
in high ee (94.2−98.2% for 7 diols; 84.2−89.9% for 6 diols)
and high yield (90−99% for 6 diols; 85−89% for 5 diols; 65%
for 1 diol). To the best of our knowledge, it is the first report of
reversing the overall enantioselectivity of cascade biocatalysis by
changing the regioselectivity in an individual reaction step,
which will help to solve the problem of lacking mirror-image
enzymes in biocatalysis.
E. coli (SSP1) and E. coli (SST1) catalyzed the trans-
dihydroxylation of either trans-aryl olefin 16a or cis-aryl olefin
17a with excellent and complementary stereoselectivity, giving
each of the four stereoisomers of 1-phenyl-1,2-propanediol 16c
in high ee and de, respectively. Both strains catalyzed the trans-
dihydroxylation of aryl cyclic olefins 18a and 19a to afford the
same trans-cyclic diols, (1R,2R)-18c and (1R,2R)-19c,
respectively, in excellent ee and de. This type of cascade
biocatalysis provides a complementary tool to Sharpless
dihydroxylation, accepting cis-alkene and offering enantioselec-
tive trans-dihydroxylation.
Preparative dihydroxylations with the resting cells of E. coli
(SSP1) or E. coli (SST1) were successfully demonstrated to
prepare five (1S)-vicinal diols and five (1R)-vincinal diols in
high ee (92.4−98.6%) with high isolated yield (70.6−85.5%).
Growing cells of E. coli (SST1) were also proven to be a good
catalyst for the enantioselective dihydroxylation of styrene 1a to
produce 120 mM (16.6 g/L) (R)-1-phenyl-1,2-ethanediol, 1c,
in 96.2% ee.
appropriate restriction enzymes (New England Biolabs). The
styA fragment was first ligated to the pRSFduet plasmid
(Novagen); the styB fragment was then ligated to the resulting
plasmid; and finally, spEH was added to the resulting construct
to give a recombinant plasmid containing three genes (styA,
styB, and spEH). By using different cloning sites on the plasmid,
three different expression cassettes, SSP1, SSP2-1, and SSP2-2,
were constructed on the pRSFduet (Figure 1a). They were
transformed into T7 Express Competent E. coli cells (New
England Biolabs) to yield E. coli (SSP1), E. coli (SSP2-1), and
E. coli (SSP2-2), respectively.
Genetic Engineering of E. coli Strains Coexpressing
Styrene Monooxygenase (SMO) and Epoxide Hydrolase
from Solanum tuberosum (StEH). The stEH gene was
synthesized with codon optimization for E. coli from Genscript
according to the sequence Genbank U02497.65 Similar to the
engineering of SMO with SpEH, the genes styA, styB, and stEH
were amplified by PCR and then cloned to the pRSFduet to
construct three expression cassettes: SST1, SST2-1, and SST2-
2, respectively (Figure 1b). The transformation of these
plasmids gave three strains (E. coli (SST1), E. coli (SST2-1),
and E. coli (SST2-2)), respectively, coexpressing SMO and
StEH.
Cell Growth and Dihydroxylation Activity of E. coli
(SSP1) or E. coli (SST1). E. coli strain E. coli (SSP1) or E. coli
(SST1) was cultured in LB medium (2 mL) containing
kanamycin (50 mg/L) at 37 °C for 7−10 h and then inoculated
into 50 mL M9 medium containing glucose (30 g/L), yeast
extract (5 g/L), and kanamycin (50 mg/L). The cells were
grown at 37 °C for 2 h to reach an OD600 of 0.6, and then
IPTG (0.5 mM) was added to induce the expression of
enzymes. The cells continued to grow for 10−12 h at 25 °C to
reach a cell density of 5−6 g cdw/L. The cells were harvested
by centrifugation (5000g, 5 min), and the cell pallets were used
as catalysts for the activity test or biotransformation.
Activity test: freshly prepared E. coli (SSP1) or E. coli (SST1)
cells were suspended to a cell density of 1.0 g cdw/L in KP
(potassium phosphate) buffer (200 mM, pH 8.0) containing
glucose (2%, w/v) and 40 μL of styrene 1a stock solution (0.5
M in ethanol) to a 2 mL system. The reaction mixture was
shaken at 250 rpm and 30 °C for 30 min. One milliliter aliquots
were taken out and mixed with 1 mL of acetonitrile to stop the
reaction. After centrifugation, the supernatant was used for
HPLC analysis of the diol product.
General Procedure for Enantioselective Dihydroxyla-
tion of Aryl Olefins 1a−22a with Resting Cells of E. coli
(SSP1) or E. coli (SST1). Freshly prepared E. coli (SSP1) or E.
coli (SST1) cells were resuspended to a cell density of 10 g
cdw/L in KP buffer (200 mM, pH 8.0) containing glucose (2%,
w/v) to a 2 mL system in a shaking flask (100 mL). Two
milliliters of n-hexadecane containing 20 mM aryl olefins 1a−
22a was added to the reaction system to form a second phase.
The reaction mixture was shaken at 250 rpm and 30 °C for 8 h,
then 200 μL aliquots of each phase were taken out at 0, 0.5, 2,
and 8 h to follow the reaction. For the organic phase, n-
hexadecane (100 μL) was separated after centrifugation, diluted
with 900 μL of n-hexane (containing 2 mM benzyl alcohol as an
internal standard), and subjected to normal phase HPLC
analysis for quantifying the olefin substrates 1a−22a and
possible epoxide intermediate. For the aqueous phase, super-
natants (100 μL) were separated after centrifugation, diluted
with 400 μL of water and 500 μL of acetonitrile (containing 2
mM benzyl alcohol as internal standard), and then used for
The cascade biocatalysis for dihydroxylation of olefins
reported here utilizes molecular oxygen as an inexpensive and
green oxidant and water as a simple reagent, thus being
sustainable. The developed catalysts show a relatively broad
substrate range for aryl olefins, high and complementary
enantioselectivity, and high activity and yield, thus being useful
for the production of several useful and valuable enantiopure
vicinal diols and deserving further development for potential
industrial application. The reported concept and methodology
on engineering efficient, enantioselective, and enantiocomple-
mentary catalysts could be extended to the development of new
biocatalysts for enantioselective trans-dihydroxylation of other
types of olefins by combining other monooxygenases and EHs.
EXPERIMENTAL SECTION
■
Genetic Engineering of E. coli Strains Coexpressing
Styrene Monooxygenase (SMO) and Epoxide Hydrolase
from Sphingomonas sp. HXN-200 (SpEH). The genes of
SMO (styA and styB) and SpEH were amplified by PCR
(Phusion DNA polymerase) from previously constructed
pSPZ1037 and the genome of Sphingomonas sp. HXN-20034
by using appropriate primers (Supporting Information). The
PCR products were subjected to double digestion with
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reversed phase HPLC analysis of the diol products 1c−22c.
The remaining aqueous phase (about 1 mL), after 8 h in the
flask, was subjected to centrifugation to remove the cells,
followed by extraction with ethyl acetate and drying over
Na2SO4. After evaporation, the residue was dissolved in 2 mL of
solvent (hexane/IPA = 9:1) for chiral HPLC analysis of the ee
and de of the diol products 1c−22c.
(R)-1-(4-Fluorophenyl)-1,2-ethanediol (R)-2c. White
solid; 0.325 g; yield, 80.7%; ee, 96.7%.
(R)-1-(3-Chlorophenyl)-1,2-ethanediol (R)-9c. Colorless
syrup; 0.304 g; yield, 70.6%; ee, 96.3%.
(1R,2S)-1-Phenyl-1,2-propanediol (1R,2S)-16c. Colorless
syrup; 0.313 g; yield, 82.3%; ee, >98%; de, 98.2%.
(1R,2R)-1-Phenyl-1,2-propanediol (1R,2R)-16c. Colorless
General Procedure for Preparation of (1S)-Vicinal
Diols (S)-1c, 2c, 5c, 9c, 12c by Enantioselective
Dihydroxylation of Aryl Olefins 1a, 2a, 5a, 9a, 12a
with Resting Cells of E. coli (SSP1). Freshly prepared E. coli
(SSP1) cells were resuspended to a cell density of 20 g cdw/L
in KP buffer (200 mM, pH 8.0) containing glucose (2%, w/v)
to a 45 mL system in a shaking flask (250 mL with tribaffle).
Five milliliters of n-hexadecane containing 2.50 mmol substrate
(0.260 g of 1a, 0.305 g of 2a, 0.295 g of 5a, 0.346 g of 9a, and
0.335 g of 12a) was added to the reaction system to form a
second phase (50 mM substrate concentration based on total
reaction volume). The reaction mixture was shaken at 250 rpm
and 30 °C, and the reaction was monitored by TLC. After 5−8
h, the substrate disappeared totally, and the reaction mixture
was then saturated with NaCl. After centrifugation, the aqueous
phase was collected and washed with 10 mL of n-hexane. The
aqueous phase was then extracted with ethyl acetate three times
(3 × 50 mL), and all the organic phases were combined. After
drying over Na2SO4, the solvents were removed by evaporation.
The crude diol products were purified by flash chromatography
on a silica gel column with n-hexane/ethyl acetate (2−1:1) as
eluent (Rf ≈ 0.3 for all diol products).
syrup; 0.300 g; yield, 78.8%; ee, 98.6%; de, >99%.
Enantioselective Dihydroxylation of Styrene 1a with
Growing Cells of E. coli (SST1). E. coli (SST1) was cultured
in LB medium (2 mL) containing kanamycin (50 mg/L) at 37
°C for 7−10 h, and then inoculated into 100 mL of M9
medium containing glucose (30 g/L), yeast extract (5 g/L), and
kanamycin (50 mg/L). The cells were grown at 30 °C for 12 h
to reach an OD600 of 15. All culture was transferred into 900
mL of sterilized modified Riesenberg medium with 15 g/L
glucose as the carbon source in a 3 L fermentor (Sartorius).
The cells were grown in the fermentor at 30 °C for 12 h to
reach an OD600 of 15−18. During the batch growth, the pH
value was maintained at 7.0 by adding 30% phosphoric acid or
25% ammonia solution, based on pH sensing, and the stirring
rate was kept constant at 1000 rpm and aeration rate was kept
constant at 1 L/min. At the end of the batch growth (12 h), the
PO started to increase, indicating glucose depletion. Fed-batch
2
growth was started by feeding a solution containing 730 g/L
glucose and 19.6 g/L MgSO4·7H2O. The feeding rate was
stepwise increased: 6.5 mL/h for 1 h, 8 mL/h for 1 h, 10 mL/h
for 1h, 13 mL/h for 1 h, then kept at 16 mL/h until the end of
the reaction. The stirring rate was stepwise increased: 1200 rpm
for 2 h and 1500 rpm for 2 h, then kept at 2000 rpm until the
end of the reaction. The aeration rate was stepwise increased:
1.2 L/min for 2 h, 1.5 L/min for 2h, then kept at 2.0 L/min
until the end of the reaction. Antifoam PEG2000 (Fluka) was
added when necessary. After fed-batch growth for 2 h, IPTG
(0.5 mM) was added to induce the expression of protein. After
fed-batch growth for 5 h, the cell density reached 20 g cdw/L,
and the biotransformation started by dropwise adding styrene
1a at the rate of 6 mL/h for 4 h and then 3 mL/h for additional
1 h. The reaction was monitored by taking a sample every hour
for analyzing the formation of diol 1c by reversed phase HPLC.
After 5 h of reaction, 120 mM (R)-1-phenyl-1,2-ethanediol was
produced in 96.2% ee.
(S)-1-Phenyl-1,2-ethanediol (S)-1c. White solid; 0.295 g;
yield: 85.5%; ee: 96.3%.
(S)-1-(4-Fluorophenyl)-1,2-ethanediol (S)-2c. White solid;
0.299 g; yield, 76.7%; ee, 96.7%.
(S)-1-(4-Methylphenyl)-1,2-ethanediol (S)-5c. White solid;
0.279 g; yield, 73.4%; ee, 92.4%.
(S)-1-(3-Chlorophenyl)-1,2-ethanediol (S)-9c. Colorless
syrup; 0.326 g; yield, 75.6%; ee, 96.5%.
(S)-1-(3-Methoxyphenyl)-1,2-ethanediol (S)-12c. Color-
less syrup; 0.358 g; yield, 85.3%; ee, 96.8%.
General Procedure for Preparation of (1R)-Vicinal
Diols (R)-1c, 2c, 9c, (1R,2S)-16c, (1R,2R)-16c by
Enantioselective Dihydroxylation of Aryl Olefins 1a,
2a, 9a, 16a, 17a with Resting Cells of E. coli (SST1).
Freshly prepared E. coli (SST1) cells were resuspended to a cell
density of 20 g cdw/L in KP buffer (200 mM, pH 8.0)
containing glucose (2%, w/v) to a 45 mL system in a shaking
flask (250 mL with tribaffle). Five milliliters of n-hexadecane
containing 2.50 mmol of substrate (0.260 g of 1a, 0.305 g of 2a,
0.346 g of 9a, 0.295 g of 16a, and 0.295 g of 17a) was added to
the reaction system in the flask. The reaction mixture was
shaken at 250 rpm and 30 °C. The reaction was monitored by
TLC. After 5−8 h, the substrate disappeared totally. The
reaction mixture was then saturated with NaCl. After
centrifugation, the aqueous phase was collected and then
washed with 10 mL of n-hexane. The aqueous phase was
extracted with ethyl acetate three times (3 × 50 mL), and all
the organic phases were combined. After drying over Na2SO4,
the solvents were removed by evaporation. The crude diol
products were then purified by flash chromatography on a silica
gel column with n-hexane/ethyl acetate (2−1:1) (Rf ≈ 0.3 for
all diol products).
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemicals, chemical synthesis, analytical methods, strains and
biochemicals, culture media, genetic engineering of recombi-
nant strains, NMR data and specific rotations of bioproducts,
procedures of other related biotransformations, time course of
representative dihydroxylation, chiral HPLC chromatograms,
1
and H NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +65-6516 8416. Fax: +65-6779 1936. E-mail: chelz@
nus.edu.sg.
Notes
(R)-1-Phenyl-1,2-ethanediol (R)-1c. White solid; 0.289 g;
yield, 83.8%; ee, 95.8%.
The authors declare no competing financial interest.
418
dx.doi.org/10.1021/cs400992z | ACS Catal. 2014, 4, 409−420

ACS Catalysis
Research Article
(30) Pham, S. Q.; Pompidor, G.; Liu, J.; Li, X. D.; Li, Z. Chem.
Commun. 2012, 48, 4618−4620.
ACKNOWLEDGMENTS
■
This work was financially supported by the Singapore-MIT
Alliance through a CPE Flagship research Program and Science
& Engineering Research Council of A*STAR, Singapore,
through a research grant (Project No. 1021010026). We also
thank Prof. Sven Panke from ETH Zurich, Switzerland, for
helpful discussion.
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