Enantioselective Cascade Biocatalysis via Epoxide Hydrolysis and Alcohol Oxidation: One-Pot Synthesis of (R)-α-Hydroxy Ketones from Meso- or Racemic Epoxides

Article abstract of DOI:10.1021/cs5016113

A new type of cascade biocatalysis was developed for one-pot enantioselective conversion of a meso- or racemic epoxide to an α-hydroxy ketone in high ee via an epoxide hydrolase-catalyzed hydrolysis of the epoxide, an alcohol dehydrogenase-catalyzed oxidation of the diol intermediate, and an enzyme-catalyzed cofactor regeneration. In vitro cascade biotransformation of meso-epoxides (cyclopentene oxide 1a, cyclohexene oxide 1b, and cycloheptene oxide 1c) was achieved with cell-free extracts containing recombinant SpEH (epoxide hydrolase from Sphingomonas sp. HXN-200), BDHA (butanediol dehydrogenase from Bacillus subtilis BGSC1A1), and LDH (lactate dehydrogenase form Bacillus subtilis) or NOX (NADH oxidase from Lactobacillus brevis DSM 20054), respectively, giving the corresponding (R)-α-hydroxycyclopentanone 3a, (R)-α-hydroxycyclohexanone 3b, and (R)-α-hydroxycycloheptanone 3c in 98-99% ee and 70-50% conversion with TTN of NAD⁺-recycling of 5500-26000. Cascade catalysis with mixed cells of Escherichia coli (SpEH) and E. coli (BDHA-NOX) converted 100-300 mM meso-epoxides 1a-1c to (R)-α-hydroxy ketones 3a-3c in 98-99% ee and 85-57% conversion. Cells of E. coli (SpEH-BDHA-NOX) coexpressing all three enzymes were also proven as good catalysts for the cascade conversion of 100-200 mM meso-epoxides 1a-1c, giving (R)-α-hydroxy ketones 3a-3c in 98-99% ee and 79-52% conversion. The cascade biocatalysis for one-pot synthesis of α-hydroxy ketone in high ee was also successfully demonstrated with a racemic epoxide (1,2,3,4-tetrahydronaphthalene-1,2-oxide 1d) as the substrate. By using two whole-cells based approaches, (R)-α-hydroxytetralone 3d was obtained in 99% ee and 49-40% conversion from 20 to 5 mM racemic epoxide 1d. Preparative cascade biotransformation of cyclohexene oxide 1b gave (R)-α-hydroxycyclohexanone 3b in 98% ee with 70% isolated yield. The developed new type of cascade biocatalysis is enantioselective, green, and often high yielding. The concept might be generally applicable to produce other useful enantiopure α-hydroxy ketones from the corresponding meso- or racemic epoxides by cascade catalysis using appropriate enzymes. (Chemical Equation Presented).

Full text of DOI:10.1021/cs5016113

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Article
Enantioselective Cascade Biocatalysis via Epoxide
Hydrolysis and Alcohol Oxidation: One-Pot Synthesis of
(R)-#-Hydroxy Ketones from Meso- or Racemic Epoxides
Jiandong Zhang, Shuke Wu, Jinchuan Wu , and Zhi Li
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/cs5016113 • Publication Date (Web): 11 Nov 2014
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Enantioselective Cascade Biocatalysis via Epoxide
Hydrolysis and Alcohol Oxidation: OneꢀPot
Synthesis of (R)ꢀαꢀHydroxy Ketones from Mesoꢀ or
Racemic Epoxides
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Jiandong Zhang,† Shuke Wu,† Jinchuan Wu,‡ and Zhi Li†,*
† Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117585
‡ Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore
627833
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ABSTRACT: A new type of cascade biocatalysis was developed for oneꢀpot enantioselective
conversion of a mesoꢀ or racemic epoxide to αꢀhydroxy ketone in high ee via an epoxide
hydrolaseꢀcatalyzed hydrolysis of the epoxide, an alcohol dehydrogenaseꢀcatalyzed oxidation of
the diol intermediate, and an enzymeꢀcatalyzed coꢀfactor regeneration. In vitro cascade
biotransformation of mesoꢀepoxides (cyclopentene oxide 1a, cyclohexene oxide 1b, and
cycloheptene oxide 1c) was achieved with cellꢀfree extracts containing recombinant SpEH
(epoxide hydrolase from Sphingomonas sp. HXNꢀ200), BDHA (butanediol dehydrogenase from
Bacillus subtilis BGSC1A1), and LDH (lactate dehydrogenase form Bacillus subtilis) or NOX
(NADH oxidase from Lactobacillus brevis DSM 20054), respectively, giving the corresponding
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(R)ꢀαꢀhydroxycyclopentanone
3a,
(R)ꢀαꢀhydroxycyclohexanone
3b,
and
(R)ꢀαꢀ
hydroxycycloheptanone 3c in 98ꢀ99% ee and 70ꢀ50% conversion with TTN of NAD+ꢀrecycling
of 5500ꢀ26000. Cascade catalysis with mixed cells of Escherichia coli (SpEH) and E. coli
(BDHAꢀNOX) converted 100ꢀ300 mM mesoꢀepoxides 1aꢀ1c to (R)ꢀαꢀhydroxy ketones 3aꢀ3c in
98ꢀ99% ee and 85ꢀ57% conversion. Cells of E. coli (SpEHꢀBDHAꢀNOX) coexpressing all three
enzymes were also proven as good catalysts for the cascade conversion of 100ꢀ200 mM mesoꢀ
epoxides 1aꢀ1c, giving (R)ꢀαꢀhydroxy ketones 3aꢀ3c in 98ꢀ99% ee and 79ꢀ52% conversion. The
cascade biocatalysis for oneꢀpot synthesis of αꢀhydroxy ketone in high ee was also successfully
demonstrated with a racemic epoxide (1,2,3,4ꢀtetrahydronaphthaleneꢀ1,2ꢀoxide 1d) as the
substrate. By using two wholeꢀcells based approaches, (R)ꢀαꢀhydroxytetralone 3d was obtained
in 99% ee and 49ꢀ40% conversion from 20ꢀ5 mM racemic epoxide 1d. Preparative cascade
biotransformation of cyclohexene oxide 1b gave (R)ꢀαꢀhydroxycyclohexanone 3b in 98% ee
with 70% isolated yield. The developed new type of cascade biocatalysis is enantioselective,
green, and often high yielding. The concept might be generally applicable to produce other
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useful enantiopure αꢀhydroxy ketones from the corresponding mesoꢀ or racemic epoxides by
cascade catalysis using appropriate enzymes.
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KEYWORDS: Biocatalysis; Cascade catalysis; Enantioselective synthesis; Epoxide hydrolase;
Alcohol dehydrogenase; αꢀHydroxy ketone.
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INTRODUCTION
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Cascade biocatalysis enables multiꢀstep reactions in one pot without the timeꢀconsuming, wasteꢀ
generating, energyꢀintensive, yieldꢀreducing, and costꢀincreasing isolation of intermediates, thus
being a green and useful tool for sustainable chemical synthesis.1ꢀ7 Many types of cascade
biocatalysis have been recently reported for organic synthesis,8ꢀ14 and enantioselective cascade
biocatalysis has also been developed for chiral synthesis, such as the deracemization of
secondary alcohols,15ꢀ16 asymmetric dihydroxylation of aryl olefins to diols,17ꢀ19 conversion of
unsaturated ketones to chiral lactones or ketones,20ꢀ21 transformation of carvenol to chiral
lactones,22 conversion of ethyl 4ꢀoxoꢀpentꢀ2ꢀenoates to chiral γ‑butyrolactones,23 amination of
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secꢀalcohols to chiral amines,24 transformation of benzaldehyde and pyruvate to
nor(pseudo)ephedrine,25 and conversion of asymmetric diketone to chiral substituted
pyrrolidines.26 Despite of the big achievement in this field, it is still necessary to develop new
types of cascade biocatalysis for many different synthetic applications.
We have been interested in developing novel cascade biocatalysis for enantioselective synthesis
of αꢀhydroxy ketones. Many enantiopure αꢀhydroxy ketones are useful templates in asymmetric
reactions and important building blocks for the synthesis of chiral chemicals and
pharmaceuticals.27ꢀ28 For instance, (R)ꢀαꢀhydroxycyclopentanone 3a and (R)ꢀαꢀ
hydroxycyclohexanone 3b are useful and valuable intermediates for chiral synthesis.29 (R)ꢀαꢀ
hydroxytetralone 3d is useful for the synthesis of 1ꢀaminoꢀ1, 2, 3, 4ꢀtetrahydronaphthalenꢀ2ꢀol, a
chiral auxiliary and ligand in asymmetric transformations.30ꢀ32 Chiral αꢀhydroxy ketones can be
prepared by using chemical methods,33ꢀ35 which often suffers from one or more drawbacks such
as the use of toxic catalyst and reagent, unsatisfied product ee, and low catalytic turnover. Chiral
αꢀhydroxy ketones can also be prepared by using enzymatic methods via resolution, which often
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encounters problem of unsatisfied enantioselectivity: acetylationꢀbased kinetic resolution
produced (S)ꢀ3d with an enantiomeric ratio (E) of 16ꢀ35;36 reductive kinetic resolution gave (R)ꢀ
3a with an E of 10;37 and oxidative kinetic resolution of transꢀcyclohexaneꢀ1,2ꢀdiol 2b with
diacetyl reductases afforded (S)ꢀ3a or (R)ꢀ3a in high ee but with low yield (< 20%).38ꢀ39 We
recently reported the oxidation of racemic transꢀvicinal diols with butanediol dehydrogenase
(BDHA) from Bacillus subtilis BGSC1A1 to give (R)ꢀαꢀhydroxy ketones 3aꢀd in high ee and 49ꢀ
50% yield with excellent enantioselectivity.40 Nevertheless, the maximum yield for kinetic
resolution is only 50%. Lyaseꢀcatalysed aldehydeꢀketone crossꢀcoupling via carboligation was
also known for the synthesis of chiral αꢀhydroxy ketones,41 however, excellent enantioselectivity
was observed only in special cases such as the reaction between pyruvate and benzaldehyde.25, 42
Here we report a new type of cascade biocatalysis for efficient oneꢀpot synthesis of enantiopure
αꢀhydroxy ketone from the corresponding easily available mesoꢀ or racemic epoxide via
enantioselective hydrolysis of the epoxide and enantioselective oxidation of the major
enantiomer of the diol intermediate (Scheme 1). The novel concept and synthetic methodology
are demonstrated with the syntheses of (R)ꢀαꢀhydroxyketones 3aꢀd as the model reactions by the
combination of an epoxide hydrolase, an alcohol dehydrogenase, and a coꢀfactor regeneration
enzyme as the efficient biocatalysts.
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RESULTS AND DISCUSSION
Selection of the enzymes and design of the biocatalytic systems for the oneꢀpot cascade
catalysis to convert mesoꢀ and racemic epoxides 1aꢀd to enantiopure αꢀhydroxy ketones 3aꢀ
d. We recently discovered a highly active epoxide hydrolase (SpEH) from Sphingomonas sp.
HXNꢀ200 as a unique bacterial EH for enantioselective hydrolysis of mesoꢀepoxides such as
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cyclopentene oxide 1a and cyclohexene oxide 1b to give the corresponding (1R, 2R)ꢀvicinal
diols such as transꢀcyclopentaneꢀ1,2ꢀdiol 2a and transꢀcyclohexaneꢀ1,2ꢀdiol 2b in 86ꢀ88% ee
and 99% yield.43,44 The EH catalyzed also the highly Rꢀenantioselective hydrolysis of racemic
epoxides such as styrene oxides to afford the corresponding (S)ꢀepoxides in >98% ee and 47ꢀ
36% yield.44ꢀ47 SpEH was thus selected as the appropriate enzyme for the hydrolysis step in the
designed cascade biotransformation of mesoꢀepoxides 1aꢀc and racemic epoxide 1,2,3,4ꢀ
tetrahydronaphthaleneꢀ1,2ꢀoxide 1d to the corresponding enantiopure αꢀhydroxy ketones 3aꢀd,
respectively (Scheme 2 and 3). Previously, we also discovered BDHA for the highly Rꢀ
enantioselective oxidation of diols 2aꢀd to give the corresponding (R)ꢀαꢀhydroxy ketones 3aꢀd in
>99% ee at 49% conversion.40 Accordingly, BDHA was chosen as the oxidation enzyme in the
cascade biocatalysis to convert the major enantiomers of the diol intermediates to the
corresponding (R)ꢀαꢀhydroxy ketones 3aꢀd. Since BDHAꢀcatalyzed oxidation depends on NAD+,
the wellꢀknown lactate dehydrogenase (LDH) from Bacillus subtilis was selected for the
regeneration of the cofactor NAD+ to achieve high productivity in the oxidation step.40 While
LDHꢀcatalyzed regeneration of NAD+ requires the addition of pyruvate as the coꢀsubstrate, it
could be more economical and greener to use NADH oxidase (NOX) for NAD+ regeneration
which requires only molecular oxygen as the coꢀsubstrate. The NOX from Lactobacillus brevis
DSM 2005448 was thus also explored as the coꢀfactorꢀregeneration enzyme in the threeꢀenzyme
cascade.
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To demonstrate the concept of using the three enzymes for the new type of cascade
biocatalysis, in vitro biocatalysis with the cellꢀfree extracts containing the three necessary
enzymes was first explored. To reduce the cost and enhance the productivity of the new cascade
biocatalysis, cells of the mixed strains expressing separately the three enzymes and cells of a
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single strain expressing all three enzymes were designed as the catalytic system, respectively,
and compared for the target reactions.
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Genetic engineering of E. coli strains expressing the individual enzyme and E. coli strains
coꢀexpressing the necessary multiple enzymes. To produce the enzymes and microbial cells for
the designed cascade reactions, E. coli (BDHA),40 E. coli (LDH),40 E. coli (SpEH),44 E. coli
(NOX), E. coli (BDHAꢀNOX), and E. coli (SpEHꢀBDHAꢀNOX) expressing the necessary
enzymes were constructed. E. coli strains were grown at 37°C in TB medium containing
appropriate antibiotics for 2 h to reach an OD600 of 0.6ꢀ0.8 and then induced by adding isopropyl
βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) at a final concentration of 0.5 mM. The cell growth was
continued at 22°C for 10ꢀ14 h. The specific activity of the cells of E. coli (NOX) harvested at 12
h was determined to be 5.2 U/mg protein by using the cellꢀfree extract for NADH oxidation with
UV detection. E. coli (BDHAꢀNOX) cells grown for 6 h to 14 h reached high specific activity
(116ꢀ129 U/g cdw) for the oxidation of racemic transꢀcyclohexaneꢀ1,2ꢀdiol 2b to αꢀ
hydroxycyclohexanone 3b (Figure S2A), which is 3ꢀfold higher than the activity of E. coli
(BDHA).40 E. coli (SpEHꢀBDHAꢀNOX) cells showed a specific activity of 34ꢀ42 U/g cdw for
the conversion of cyclohexene oxide 1b to αꢀhydroxycyclohexanone 3b after growth for 6ꢀ14
(Figure S2B).
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The SDSꢀPAGE of the cellꢀfree extracts of the E. coli cells harvested at 12 h (after 10 h
induction) was shown in Figure S2C. The protein bands of the coꢀexpressed SpEH, BDHA, and
NOX in E. coli (SpEHꢀBDHAꢀNOX) (Lane 1), the coꢀexpressed BDHA and NOX in E. coli
(BDHAꢀNOX) (Lane 2), the expressed NOX in E. coli (NOX) (lane 3), the expressed SpEH in E.
coli (SpEH) (lane 4), and the expressed BDHA in E. coli (BDHA) (lane 5) were clearly visible.
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Cascade biocatalysis with SpEH, BDHA, and LDH or with SpEH, BDHA, and NOX in
vitro for oneꢀpot conversion of mesoꢀepoxide 1aꢀc to (R)ꢀαꢀhydroxyketones 3aꢀc. Cellꢀfree
extracts of E. coli (SpEH), E. coli (BDHA), E. coli (LDH), and E. coli (NOX) were prepared
from cells harvested at late exponential growing phase at 12ꢀ14 h, respectively, and they were
used for in vitro cascade biocatalysis to demonstrate the concept (Scheme 2). To facilitate the
adjustment of the ratios among different enzymes for accomplishing the cascade reaction, the
cellꢀfree extracts of the E. coli strains were lyophilized to give the corresponding enzyme
powders, respectively. SpEH and BDHA were used at a ratio of 1:2 in the presence of 0.002 mM
NADH for biotransformation of cyclopentene oxide 1a, and only 4% conversion to αꢀ
hydroxycyclopentanone 3a was obtained at 12 h (Table 1, entry 1). The conversion was greatly
improved by the addition of LDH as a coꢀfactorꢀregeneration enzyme in the catalytic system.
Combining SpEH, BDHA, and LDH at 3ꢀ5:10:5 in the presence of 0.002ꢀ0.004 mM NADH and
200 mM pyruvate gave rise to the conversion of mesoꢀepoxides 1aꢀc (25ꢀ100 mM) to (R)ꢀ
hydroxy ketones (R)ꢀ3aꢀc in excellent ee (98ꢀ99%) and good conversion (50ꢀ65%) after 9ꢀ12 h
reaction (Table 1, entry 2ꢀ4). TTN of 5500ꢀ26000 for NAD+ regeneration was achieved, which is
high enough for economical application of NAD+. However, the regeneration of NAD+ requires
the excessive of pyruvate as coꢀsubstrate thus generating additional cost and waste. The use of
NOX as the coꢀfactor regeneration enzyme was then examined. By using a catalytic system
containing SpEH, BDHA, and NOX at 3ꢀ5:10ꢀ12:5 in the presence of 0.002ꢀ0.004 mM NADH,
(R)ꢀhydroxy ketones 3aꢀc were obtained in excellent ee (98ꢀ99%) and good conversion (58ꢀ
70%) from mesoꢀepoxides 1aꢀc (20ꢀ100 mM) after 12ꢀ18 h reactions (Table 1, entry 5ꢀ7). In
such a system, the regeneration of NAD+ requires only molecular oxygen as coꢀsubstrate and
gave only water as byproduct, and the TTN for the regeneration of NAD+ reached 6200ꢀ14000
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which is also high enough for practical application of the cofactor. Considering the higher atom
efficiency and less waste generation, NOXꢀcatalyzed regeneration of NAD+ is chosen for further
development of the cascade biocatalysis.
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The in vitro biotransformations results successfully demonstrated the new type of cascade
biocatalysis and novel methodology for oneꢀpot production of (R)ꢀhydroxy ketones from the
corresponding mesoꢀepoxides via enantioselective epoxide hydrolysis and enantioselective
oxidation of the diol intermediate. In the first step, SpEH catalysed the enantioselective
hydrolysis of mesoꢀepoxides 1aꢀb to give the corresponding (R, R)ꢀdiols 2aꢀb in 85ꢀ86% ee. In
the second step, BDHA catalysed the highly enantioselective oxidation of the major enantiomer
(R, R)ꢀ2aꢀb to afford the corresponding (R)ꢀhydroxy ketones 3aꢀb in 98ꢀ99% ee. In the case of
conversion of cycloheptene oxide 1c to (R)ꢀhydroxycycloheptanone 3c, hydrolysis with SpEH
gave (R, R)ꢀtransꢀcycloheptaneꢀ1,2ꢀdiol 2c in only 19% ee. Nevertheless, the highly selective
oxidation of (R, R)ꢀ2c with BDHA gave rise to the formation of (R)ꢀhydroxycycloheptanone 3c
still in excellent ee (99%). In comparison, chemical conversion of epoxides to αꢀhydroxy ketones
with strong acids gave racemic product together with several byproducts.49ꢀ51
Cascade biocatalysis with the mixtures of resting cells of E. coli (SpEH) and E. coli
(BDHAꢀNOX) for oneꢀpot conversion of mesoꢀepoxide 1aꢀc to (R)ꢀαꢀhydroxyketones 3aꢀc.
The use of wholeꢀcell biocatalysts is more economic for coꢀfactor dependent biotransformation
than the use of free enzymes, since the cells can be easily produced in large amount and at low
cost, the intact cells can help to maintain enzyme stability, and the cofactor NAD+/NADH in
cells can be used for target reaction with coꢀfactor recycling. To develop wholeꢀcell system for
the cascade biocatalysis, the resting cells of E. coli (BDHAꢀNOX) were first examined for the
oxidation reaction in the second step. The cells showed a specific activity of 115, 150, 143 U/g
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cdw for the oxidation of racemic transꢀcyclic diols 2aꢀc to (R)ꢀαꢀhydroxyketones 3aꢀb,
respectively, being 3ꢀfold higher than the cells of E. coli (BDHA).40 The enhanced activity is
possibly due to the regeneration of intracelluer cofactor NAD+. Biooxidation of racemic transꢀ
cyclic diol 2aꢀc (100 mM) with resting cells of E. coli (BDHAꢀNOX) gave (R)ꢀαꢀ
hydroxyketones 3aꢀc in 97ꢀ99% ee and 49ꢀ50% conversion, being also more productive than that
with resting cells of E. coli (BDHA).40 On the other hand, the resting cells of E. coli (SpEH)
were also highly active for the hydrolysis reaction in the first step of the cascade, converting
mesoꢀepoxides 1aꢀc (100 mM) to the corresponding diols in 99% within only 1ꢀ2 h. As same as
the free SpEH, the wholeꢀcells gave the hydrolysis products (R, R)ꢀ2aꢀb in 85ꢀ86% ee and (R,
R)ꢀ2c in 19% ee, respectively.
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Based on the different specific activities of the two bacterial cells, the resting cells of E. coli
(SpEH) and E. coli (BDHAꢀNOX) were mixed at a ratio of 4:10ꢀ16 to achieve efficient cascade
biocatalysis. To solve the solubility and toxicity problem of the epoxides, an aqueousꢀorganic
twoꢀphase system consisting of Trisꢀbuffer and hexadecane at 1:1 (v/v) was used as the reaction
medium in which the hydrophobic organic phase acts as a reservoir for the toxic epoxides. As
shown in Table 2, combining E. coli (SpEH) cells at 4 g cdw/L and E. coli (BDHAꢀNOX) cells
at 12 g cdw/L was able to produce (R)ꢀαꢀhydroxycyclohexanone 3b in 98.0% ee with 85%
conversion from 100 mM cyclohexene oxide 1b (Table 2, entry 2). The time course of this
reaction was shown in Figure 1. At 1 h, epoxide 1b was totally converted to transꢀcyclohexaneꢀ
1,2ꢀdiol 2b due to the high activity of SpEH, 72 mM diol 2b remained in the reaction mixture,
and the rest of diol 2b was converted to 26 mM (R)ꢀαꢀhydroxycyclohexanone 3b in 99% ee by
BDHA coupled with NOX. From 1 h to 6 h, the concentration of (R)ꢀ3b increased linearly with
the linear decrease of the concentration of 2b. At 9 h, 85 mM (R)ꢀ3b was produced in 98% ee,
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and 13 mM 2b remained in the reaction mixture. To achieve even higher product concentration,
200ꢀ300 mM cyclohexene oxide 1b was used for the cascade catalysis with the cell mixtures of
E. coli (SpEH) at 4 g cdw/L and E. coli (BDHAꢀNOX) at 16 g cdw/L as catalysts. These gave
(R)ꢀαꢀhydroxyꢀcyclohexanone 3b in 98% ee with 78ꢀ70% conversion.
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The generality of using the mixtures of the cells containing three necessary enzymes for
performing the cascade reaction was demonstrated by using other mesoꢀepoxides 1a and 1c as
the substrates. As shown in Table 2 (entry 1 and entry 5), conversion of cyclopentene oxide 1a
(100 mM) and cycloheptene oxide 1c (200 mM) with a mixture of 4 g cdw/L of E. coli (SpEH)
cells and 10ꢀ13 g cdw/L of E. coli (BDHAꢀNOX) cells gave (R)ꢀαꢀhydroxycyclopentanone 3a in
98% ee with 84% conversion and (R)ꢀαꢀhydroxycycloheptanone 3c in 99% ee with 57%
conversion, respectively. In comparison with the cascade biocatalysis using free enzymes, the
use of cell mixtures afforded high product concentration and costꢀeffectiveness (considering the
cost and availability of catalyst and cofactor) with the potential for industrial application.
Cascade biocatalysis with resting cells of E. coli (SpEHꢀBDHAꢀNOX) for oneꢀpot
conversion of mesoꢀepoxide 1aꢀc to (R)ꢀαꢀhydroxyketones 3aꢀc. The use of single recombinant
strain coꢀexpressing all necessary enzymes for the cascade biocatalysis could avoid the cell
cultivation of multiple strains and reduce the total cell density for the cascade biocatalysis.
Resting cells of E. coli (SpEHꢀBDHAꢀNOX) were thus used for the oneꢀpot conversion of mesoꢀ
epoxide 1aꢀc to (R)ꢀhydroxy ketones 3aꢀc (Scheme 2). As shown in Table 2 (entry 6ꢀ9),
reactions of 100ꢀ200 mM epoxide 1b with E. coli (SpEHꢀBDHAꢀNOX) at 12ꢀ16 g cdw/L
afforded (R)ꢀαꢀhydroxycyclohexanone 3b in 98% ee and 71ꢀ60% conversion. Reaction of
epoxides 1a and 1c (100 mM) with the same catalysts at 12 g cdw/L gave (R)ꢀαꢀ
hydroxycyclopentanone 3a in 98% ee and 79% conversion and (R)ꢀαꢀhydroxycycloheptanone 3c
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in 99% ee and 52% conversion, respectively. These results proved that the use of cells of a single
microorganism coꢀexpressing three necessary enzymes as catalysts worked well for the desired
cascade biocatalysis. In comparison with the use of the cell mixtures of E. coli (SpEH) and E.
coli (BDHAꢀNOX), similar product yields were achieved at lower cell density with E. coli
(SpEHꢀBDHAꢀNOX) as catalysts. On the other hand, the use of mixed microorganisms is of
advantages regarding the easy adjustment of appropriate ratio of different enzymes.
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Cascade biocatalysis for oneꢀpot conversion of racemic epoxide 1d to (R)ꢀαꢀ
hydroxyketones 3d with the mixtures of resting cells of E. coli (SpEH) and E. coli (BDHAꢀ
NOX) or with resting cells of E. coli (SpEHꢀBDHAꢀNOX). To further prove the generality of
the new cascade biocatalysis, racemic 1,2,3,4ꢀtetrahydronaphthaleneꢀ1,2ꢀoxide 1d was selected
as a substrate for oneꢀpot synthesis of αꢀhydroxyketone with resting cells of E. coli (SpEH). This
reaction was not very enantioselective, but it could quickly reach 100% conversion to produce
racemic transꢀ1,2ꢀdihydroxyꢀ1,2,3,4ꢀtetrahydronaphthalene 2d. On the other hand, the oxidation
of racemic transꢀdiol 2d with resting cells of E. coli (BDHAꢀNOX) was highly enantioselective,
providing (R)ꢀαꢀhydroxytetralone 3d in >99% ee at 49% conversion. The combining of cells of
E. coli (SpEH) and E. coli (BDHAꢀNOX) for the cascade conversion of 5ꢀ20 mM racemic
epoxide 1d gave (R)ꢀ3d in 99% ee with 49ꢀ44% conversion (Table 3, entry 1ꢀ2). Alternatively,
the use of resting cells of E. coli (SpEHꢀBDHAꢀNOX) alone converted 10 mM racemic 1d to
(R)ꢀαꢀhydroxytetralone 3d in 99% ee and 40% conversion (Table 3, entry 3). All these results
demonstrated the generality of using the cascade biocatalysis for oneꢀpot synthesis of
enantiopure αꢀhydroxyketones from the corresponding epoxides, either mesoꢀepoxides or
racemic epoxides. The low yield in the cascade conversion of a racemic epoxide to an
enantiopure αꢀhydroxyketone might be improved by using an enantioconvergent EH52ꢀ53 as the
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first enzyme in the cascade reaction to convert a racemic epoxide into an enantioꢀenriched or
pure diol with high conversion.
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Preparation of (R)ꢀαꢀhydroxycyclohexanone 3b from cyclohexene oxide 1b by oneꢀpot
cascade biocatalysis with the mixtures of the resting cells of E. coli (SpEH) and E. coli
(BDHAꢀNOX). The applicability of this new type of cascade biocatalysis for preparative
biotransformation was examined on a 100 mLꢀscale biotransformation. Reaction of cyclohexene
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o
oxide 1b (100 mM, 500 mg) was carried out at 30 C for 9 h in a twoꢀliquid phase system
consisting of 50 mL hexadecane and 50 mL aqueous buffer containing the resting cells of E. coli
(SpEH) (4 g cdw/L) and E. coli (BDHAꢀNOX) (12 g cdw/L). Although some transꢀ
cyclohexaneneꢀ1,2ꢀdiol 2b still remained in the reaction mixtures, simple workꢀup and
subsequent purification by flash chromatography gave pure (R)ꢀαꢀhydroxycyclohexanone 3b in
98% ee with 70% isolated yield (350 mg). This example demonstrated the potential of using the
new type of cascade biocatalysis for facile, green, and highꢀyielding synthesis of enantiopure αꢀ
hydroxycyclohexanone from the corresponding easily available epoxide.
CONCLUSION
A new type of cascade biocatalysis was successfully developed for oneꢀpot synthesis of useful
and valuable αꢀhydroxy ketones in high ee from the corresponding easily available mesoꢀ or
racemic epoxides via enantioselective epoxide hydrolysis and enantioselective oxidation of the
diol intermediate. Efficient cascade biocatalysis was achieved by the combination of an epoxide
hydrolase, an alcohol dehydrogenase, and a coꢀfactor regeneration enzyme as the catalysts. The
novel concept and synthetic methodology were successfully demonstrated by in vitro
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biocatalysis with the mixtures of recombinant enzymes SpEH, BDHA, and LDH or NOX, in the
presence of trace amount of NADH, to convert several cyclic mesoꢀepoxides 1aꢀc to the
corresponding (R)ꢀαꢀhydroxy ketones 3aꢀc in 98ꢀ99% ee and 70ꢀ50% conversion with a TTN of
5500ꢀ26000 for NAD+ recycling. In comparison with LDH, NOX catalyzed the regeneration of
NAD+ by using molecular oxygen as coꢀsubstrate with water as coꢀproduct, thus being more
economic and sustainable. Wholeꢀcell based cascade biocatalysis was achieved by using the cell
mixtures of E. coli (SpEH) and E. coli (BDHAꢀNOX) at the optimized ratio in a twoꢀliquid phase
system to convert 100ꢀ300 mM mesoꢀepoxide 1aꢀc to (R)ꢀαꢀhydroxy ketones 3aꢀc in 98ꢀ99% ee
and 85ꢀ57% conversion. Moreover, the cells of E. coli (SpEHꢀBDHAꢀNOX) coexpressing all
three enzymes were also good catalysts for the cascade conversion, converting 79ꢀ52% of 100ꢀ
200 mM mesoꢀepoxide 1aꢀc to (R)ꢀαꢀhydroxy ketones 3aꢀc in 98ꢀ99% ee. The generality of the
cascade biocatalysis was further demonstrated with these wholeꢀcell approaches to convert 5ꢀ20
mM racemic epoxide 1d to (R)ꢀαꢀhydroxytetralone 3d in 99% ee with 49ꢀ40% conversion.
Preparative biotransformation was demonstrated on a 100 mLꢀscale with mixed cells of E. coli
(SpEH) and E. coli (BDHAꢀNOX) to give (R)ꢀαꢀhydroxycyclohexanone 3b in 98% ee and 70%
isolated yield from cyclohexene oxide 1b (100 mM). The new type of cascade biocatalysis might
be generally applicable to prepare other useful enantiopure αꢀhydroxyketones from the
corresponding mesoꢀ or racemic epoxides by selecting and combining the appropriate enzymes
in the similar way.
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Engineering of E. coli (BDHA), E. coli (LDH), E. coli (SpEH), E. coli (NOX), E. coli
(BDHAꢀNOX), and E. coli (SpEHꢀBDHAꢀNOX). E. coli (BDHA) and E. coli (LDH) were
constructed in our previous research.40
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E. coli (SpEH): epoxide hydrolase (SpEH) gene44 was amplified via PCR with primers of
SpEHꢀF: CGC GGATCC G ATG ATG AAC GTC GAA CAT ATC CG and SpEHꢀR: CCC
AAGCTT TCA AAG ATC CAT CTG TGC AAA GGC by using genome DNA from
Sphingomonas sp. HXNꢀ200 as the template. The PCR amplifications were performed with Pfu
DNA polymerase (New England Biolabs Inc.). The resulting 1146 bp fragment was digested
with BamHI and HindIII and then ligated into pETduetꢀ1 which was then digested with the same
restriction enzymes to generate the construct pETduetꢀSpEH. The resulting construct was then
transformed into E. coli T7 competent cells and plated on LB plates containing ampicillin (100
µg/mL). The transformed strain was abbreviated as E. coli (SpEH).
E. coli (NOX): NADH oxidase (NOX) gene from Lactobacillus brevis DSM 2005448 was
synthesized by Genscript Corp (Piscataway, NJ). The gene was PCR amplified further using
forward primer NOXꢀF: GGA AGATCT C ATG AAA GTC ACA GTT GTT GGT TGT AC and
reverse primer NOXꢀR: CCG CTCGAG TTA AGC GTT AAC TGA TTG GGC AAC T. The
amplified gene was digested with BglII and XhoI and ligated into pETduet vector. The resulting
construct was then transformed into E. coli T7 competent cells and plated on LB plates
containing 100 µg/mL ampicillin. The transformed strain was abbreviated as E. coli (NOX).
E. coli (BDHAꢀNOX): The constructed plasmids pET28aꢀBDHA40 and pETduetꢀNOX with
the same origin of replication and different antibiotic selection were transformed into E. coli T7
competent cells simultaneously and plated on LB plates containing ampicillin (100 µg/mL) and
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kanamycin (50 µg/mL). The transformed strain coꢀexpressing BDHA and NOX was abbreviated
as E. coli (BDHAꢀNOX).
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E. coli (SpEHꢀBDHAꢀNOX): pETduetꢀSpEH was first digested with BglII and XhoI, and the
NOX gene (digested with BglII and XhoI) was ligated into pETduetꢀSpEH vector, generating the
construct pETduetꢀSpEHꢀNOX. The constructed plasmids pET28aꢀBDHA40 and pETduetꢀSpEHꢀ
NOX with the same origin of replication and different antibiotic selection were transformed into
E. coli T7 competent cells simultaneously and plated on LB plates containing ampicillin (100
µg/mL) and kanamycin (50 µg/mL). The transformed strain coꢀexpressing BDHA, SpEH and
NOX was abbreviated as E. coli (SpEHꢀBDHAꢀNOX).
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Cell growth and activity of E. coli (SpEH), E. coli (BDHA), E. coli (LDH), E. coli (NOX),
E. coli (BDHAꢀNOX), and E. coli (SpEHꢀBDHAꢀNOX). The recombinant E. coli strains were
grown, respectively, overnight at 37°C in LB medium (3 mL in 20 mL tube) containing
appropriate antibiotics (100 µg/mL ampicillin, or 50 µg/mL kanamycin, or both) as indicated
above. 1 mL seed culture was transferred to 50 mL TB medium containing appropriate
antibiotics in 250 mL flask. The cells were grown for 2 h to reach an OD600 of 0.6ꢀ0.8 and then
induced with isopropyl βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) (final concentration of 0.5 mM).
Cell growth was continued at 22°C and 250 rpm for 10ꢀ12 h. Cells were harvested by
centrifugation at 8,500×g and 4°C for 10 min, washed twice with Tris buffer (100 mM, pH 7.5),
and resuspended in the same buffer as resting cells for activity test and biotransformation.
The activity tests for E. coli (SpEH), E. coli (BDHA), and E. coli (LDH) were performed as
described before.44,40 The specific activity of E. coli (BDHAꢀNOX) was determined by
performing the biotransformation of racemic cyclohexaneꢀdiols 2b (50 mM) to (R)ꢀαꢀ
hydroxycyclohexanone 3b with the resting cells (2 g cdw/L) in 10 mL Tris buffer (pH 7.5, 100
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mM) at 30°C and 250 rpm for 10 min and quantifying the product concentration by GC analysis.
The specific activity of E. coli (SpEHꢀBDHAꢀNOX) was determined by performing the
biotransformation of cyclohexene oxide 1b (50 mM) to (R)ꢀαꢀhydroxycyclohexanone 3b with the
resting cells (2 g cdw/L) in 10 mL Tris buffer (pH 7.5, 100 mM) at 30°C and 250 rpm for 10
min, and quantifying the product concentration by GC analysis. Analytic samples for these
assays were prepared by removing the cells by centrifugation, saturating with NaCl, extracting
with ethyl acetate containing 5 mM phenylacetone as an internal standard (1:1), and drying over
Na2SO4. The special activities were expressed in U/g cdw, and all experiments were performed
in duplicate.
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The specific activity of E. coli (NOX) was performed by using the cellꢀfree extract. The
harvested cell pellets were reꢀsuspended in 20 mL Tris buffer (pH 7.5, 100 mM) to an OD600 of
25 and passed through a homogenizer (one time, at 21 bar) to break the cells. The cell lysate was
centrifuged at 15,000 × g and 4oC for 30 min, and the supernatant (cellꢀfree extract) was used to
determine the activity of NOX. The protein concentration in the cellꢀfree extracts was
determined by the Bradford method. The activity assay was performed with a mixture of 989 µL
Tris buffer (pH 7.5, 100 mM), 10 µL NADH (20 mM) in distilled water, and 1 µL enzyme
solution (5 g protein/L) in a cuvette. The UV absorbance at 340 nm was followed to monitor the
decrease of NADH concentration by the oxidation with NOX. One unit of enzyme activity was
defined as 1 µmol NADH decreased per minute. The special activity was expressed in U/g
protein, and all experiments were performed in duplicate.
General procedure for enantioselective cascade conversions of mesoꢀepoxides 1aꢀc to (R)ꢀ
αꢀhydroxy ketones 3aꢀc with the mixture of lyophilized cellꢀfree extract of E. coli (SpEH),
E. coli (BDHA), and E. coli (LDH) or E. coli (NOX). The freshly prepared cells of E. coli
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(SpEH), E. coli (BDHA), E. coli (LDH), and E. coli (NOX) were resuspended in deionized (DI)
water to a cell density of 20 g cdw/L, respectively. The cell suspension was passed through a
Constant Cell Disruption System at 20 KPSi to break the cells, and the mixture was
ultracentrifuged at 15,000 g at 4oC for 30 min to remove the cell debris. The protein
concentration of the cellꢀfree extracts was determined by Bradford method. The cellꢀfree extracts
were frozen at ꢀ80oC overnight, followed by lyophilization for 48 h to get the lyophilized enzyme
powders.
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The lyophilized powders of the cell freeꢀextracts of E. coli (SpEH), E. coli (BDHA), and E.
coli (LDH) or E. coli (NOX) were mixed at different protein concentration (3ꢀ5 g/L, 10ꢀ12 g/L,
and 5 g/L, respectively) in 10 mL 100 mM Tris buffer (pH 7.5), followed by the addition of
mesoꢀepoxides 1aꢀc (20ꢀ100 mM), NADH (0.002ꢀ0.004 mM), and coꢀsolvent DMSO (5% v/v).
In the case of using lyophilized power of the cell freeꢀextracts of E. coli (LDH), pyruvate (200
mM) was also added as the coꢀsubstrate. The mixtures were shaken at 250 rpm and 30°C. 300 µL
aliquots were taken out at different time points, saturated with NaCl, and extracted with ethyl
acetate containing 5 mM phenylacetone as an internal standard (1:1). The organic phase was
collected and dried over Na2SO4. After filtration, the samples were used for GC analysis to
determine the yield and ee of (R)ꢀαꢀhydroxy ketones 3aꢀc.
General procedure for enantioselective oxidation of racemic diols 2aꢀd to (R)ꢀαꢀhydroxy
ketones 3aꢀd with resting cells of E. coli (BDHAꢀNOX). The freshly prepared E. coli (BDHAꢀ
NOX) cells were reꢀsuspended in 10 mL 100 mM Tris buffer (pH 7.5) to a cell density of 10ꢀ16
g cdw/L, racemic diols 2aꢀd (0.2ꢀ2.0 mmol) was added, and the mixtures were shaken at 250
rpm and 30°C. For the biotransformation of 2aꢀc, 300 µL aliquots were taken out from the
reaction mixtures at different time points for GC analysis of the ee and concentration of diols 2aꢀ
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c and αꢀhydroxy ketones 3aꢀc. Analytic samples were prepared by removing the cells by
centrifugation, saturating with NaCl, extracting with ethyl acetate containing 5 mM
phenylacetone as an internal standard (1:1), and drying over Na2SO4. For the biotransformation
of 2d, 100 µL aliquots were taken out from the reaction mixtures at different time points, mixed
with 400 µL ACN containing 2 mM benzylacetone as an internal standard, and used for HPLC
analysis to determine the concentration of diol 2d and αꢀhydroxy ketone 3d. For analyzing the ee
of 2d and 3d, 200 µL aliquots were taken out and centrifuged to remove the cells, followed by
extraction with 200 µL chloroform, evaporation, and dissolving the residues in 200 ꢁL isopropyl
alcohol for chiral HPLC analysis.
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General procedure for enantioselective cascade conversions of epoxides 1aꢀd to (R)ꢀαꢀ
hydroxy ketones 3aꢀd with the mixtures of resting cells of E. coli (SpEH) and E. coli
(BDHAꢀNOX). The freshly prepared cells of E. coli (SpEH) and E. coli (BDHAꢀNOX) were
mixed in 10 mL 100 mM Tris buffer (pH 7.5) to a cell density of 4 g cdw/L and 10ꢀ16 g cdw/L,
respectively. 10 mL hexadecane containing epoxides 1aꢀd (0.1ꢀ2.0 mmol) was added. The
mixture was incubated at 250 rpm and 30°C. For 1aꢀc, 300 µL aqueous phases were taken out at
different time points for analysis. Analytic samples were prepared by removal of the cells via
centrifugation, saturation with NaCl, extraction with ethyl acetate (1:1) containing 5 mM
phenylacetone as an internal standard, and drying the organic phase over Na2SO4. The samples
were analysed by GC to quantify the ee and concentration of diols 2aꢀc and αꢀhydroxy ketones
3aꢀc. For 1d, 10 µL organic and 100 µL aqueous phases were taken out separately at different
time points and diluted with 490 µL and 400 µL ACN containing 2 mM benzylacetone as an
internal standard, respectively. The samples were analysed by HPLC to determine the
concentration of diol 2d and αꢀhydroxy ketone 3d. The concentration of each compound was
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calculated as the total concentration in aqueous and organic phase. For analyzing the ee of (R)ꢀ
3d, 200 µL aliquots were taken out from the reaction mixture, the cells were removed by
centrifugation, the product was extracted with 200 µL chloroform, the solvent was evaporated,
and the residue was reꢀdissolved in 200 ꢁL isopropyl alcohol for chiral HPLC analysis.
General procedure for enantioselective cascade conversions of epoxides 1aꢀd to (R)ꢀαꢀ
hydroxy ketones 3aꢀd with the resting cells of E. coli (SpEHꢀBDHAꢀNOX). The freshly
prepared E. coli (SpEHꢀBDHAꢀNOX) cells were resuspended in 10 mL 100 mM Tris buffer (pH
7.5) to a cell density of 12ꢀ16 g cdw/L. 10 mL hexadecane containing epoxides 1aꢀd (0.1ꢀ2.0
mmol) was added. The mixture was incubated at 250 rpm and 30°C. Samples were taken out at
different time points to prepare analytic samples for the determination of the product
concentration and ee, as described above.
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Preparation of (R)ꢀαꢀhydroxycyclohexanone 3b from cyclohexene oxide 1b by cascade
biocatalysis with the mixtures of resting cells of E. coli (SpEH) and E. coli (BDHAꢀNOX).
The freshly prepared cells of E. coli (SpEH) and E. coli (BDHAꢀNOX) were resuspended in 50
mL 100 mM Tris buffer (pH 7.5) to a cell density of 4 g cdw/L and 12 g cdw/L, respectively. 50
mL hexadecane containing cyclohexene oxide 1b (100 mM, 500 mg) was added. The mixtures
were shaken at 250 rpm and 30°C for 9 h. The reaction mixture was centrifuged to remove the
cells. Aqueous phase was separated, saturated with NaCl, and extracted with ethyl acetate for
three times (3 × 50 mL). All the organic phases were combined and then dried over Na2SO4.
After filtration, the organic solvent was removed by evaporation at reduced pressure. The crude
products were purified by flash chromatography on a silica gel column with nꢀhexane:ethyl
acetate of 50:50 (Rf = 0.3) to give (R)ꢀ3b as a colourless oil in 70.0% yield (350 mg) and 98% ee.
1
[α]D25 +20.6 (c 1.0, CHCl3); Lit.14: [α]D20 +20.8 (c 0.65, CHCl3, >99% ee). HꢀNMR (400 MHz,
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CDCl3) δ: 1.44ꢀ1.78 (m, 3H), 1.85ꢀ1.93 (m, 1H), 2.08ꢀ2.15 (m, 1H), 2.32ꢀ2.41 (m, 1 H), 2.43ꢀ
2.50 (m, 1 H), 2.54ꢀ2.60 (m, 1H), 3.64 (d, J = 3.2 Hz, 1H), 4.10ꢀ4.15 (m, 1H).
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ASSOCIATED CONTENT
Supporting Information.
Chemicals, biochemicals, and strains; analytical methods; specific activity of NOX for the cellꢀ
free extract; specific activity of SpEH, BDHA, LDH, and NOX for the lyophilized cellꢀfree
extracts; procedure for the hydrolysis of epoxides with E. coli (SpEH); data for the oxidation of
diols with E. coli (BDHA) and E. coli (BDHAꢀNOX); chiral GC and HPLC chromatograms; and
1H NMR spectrum. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Phone: +65ꢀ65168416. Fax: +65ꢀ67791936. Eꢀmail: chelz@nus.edu.sg.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Science & Engineering Research Council of A*STAR, Singapore, for the financial
support through a research grant (project No. 1021010026).
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SCHEMES
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Scheme 1. Oneꢀpot synthesis of enantiopure αꢀhydroxy ketones from mesoꢀ or racemic epoxides
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via cascade biocatalysis.
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Scheme 2. Oneꢀpot synthesis of (R)ꢀαꢀhydroxy ketones 3aꢀc from mesoꢀepoxides 1aꢀc via
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Scheme 3. Oneꢀpot synthesis of (R)ꢀαꢀhydroxy ketone 3d from racemic epoxide 1d via cascade
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