Development of a Practical, Biocatalytic Synthesis of tert-Butyl (R)-3-Hydroxyl-5-hexenoate: A Key Intermediate to the Statin Side Chain

Article abstract of DOI:10.1021/acs.oprd.0c00320

The HMG-CoA reductase inhibitors, statins, are one of the most effective and bestselling cholesterol-lowering drugs. The use of statins has greatly extended people's lives and improved the quality of their life. Development of a more efficient, stereoselective, and sustainable synthesis of statins is continuingly of utmost importance. In the present study, through screening of ketoreductases (KREDs) and reaction optimization, we have successfully performed a highly stereoselective reduction of ketoester 1a catalyzed by KRED-06 at a pilot-plant scale without the addition of exogenous NADP+, generating 3.21 kg of enantiomerically pure tert-butyl (R)-3-hydroxyl-5-hexenoate ((R)-2a) (96.2% yield, >99.9% enantiomeric excess (ee)). This newly developed biocatalytic process alleviates the cryogenic conditions (-40 °C) employed in our first-generation synthesis of (R)-2a using NaBH4 and (l)-tartaric acid. Coupled with our previously established synthesis of bromocarbonate 3a via a one-pot diastereoselective carboxylation/bromocyclization of (R)-2a, we have developed an innovative, practical synthesis route to statin side chain, possessing great potential to be implemented into industrial production of statins.

Full text of DOI:10.1021/acs.oprd.0c00320

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Full Paper
Development of a Practical, Biocatalytic Synthesis of Tert-Butyl
(R)-3-Hydroxyl-5-Hexenoate: a Key Intermediate to Statin Side Chain
Chen Hu, Minjie Liu, Xiaoping Yue, Zedu Huang, and Fener Chen
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00320 • Publication Date (Web): 21 Aug 2020
Downloaded from pubs.acs.org on August 22, 2020
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Development of a Practical, Biocatalytic
Synthesis of Tert-Butyl (R)-3-Hydroxyl-5-
Hexenoate: a Key Intermediate to Statin Side
Chain
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Chen Hu, [a,b]Minjie Liu,[a,b]Xiaoping Yue, [c]Zedu Huang,* [a,b] and Fener Chen*[a,b]
[a] Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of
Chemistry, Fudan University, 220 Handan Road, Shanghai, 200433, P. R. China
[b] Shanghai Engineering Research Center of Industrial Asymmetric Catalysis of Chiral Drugs,
220 Handan Road, Shanghai, 200433, P. R. China
[c] West China School of Pharmacy, Sichuan University, Chengdu, 610041, P. R. China
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ABSTRACT: The HMG-CoA reductase inhibitors, statins, are one of the most effective and
best-selling cholesterol-lowering drugs. The use of statins has greatly extended people’s lives and
improved the quality of their life. Development of a more efficient, stereoselective, and sustainable
synthesis of statins is continuingly of utmost importance. In the present study, through screening
of ketoreductases (KREDs) and reaction optimization, we have successfully performed a highly
stereoselective reduction of ketoester 1a catalyzed by KRED-06 at a pilot-plant-scale without the
addition of exogenous NADP+, generating 3.21 kg of enantiomerically pure tert-butyl (R)-3-
hydroxyl-5-hexenoate ((R)-2a) (96.2% yield, >99.9% ee). This newly developed biocatalytic
process alleviates the cryogenic conditions (-40 ℃) employed in our first-generation synthesis of
(R)-2a using NaBH4 and (L)-tartaric acid. Coupled with our previously established synthesis of
bromocarbonate 3a via a one-pot diastereoselective carboxylation/bromocyclization of (R)-2a, we
have developed an innovative, practical synthesis route to statin side chain, possessing great
potential to be implemented into industrial production of statins.
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KEYWORDS: asymmetric catalysis, ketoreductase, statin, tert-butyl (R)-3-hydroxyl-5-
hexenoate, pilot-plant-scale synthesis
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Introduction
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Since their introduction to market in 1987, statins (Scheme 1A), the inhibitors of 3-
hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), have developed as one
of the top selling families of drugs prescribed for lowering cholesterol, hence preventing
cardiovascular diseases such as hypercholesterolemia, and coronary heart disease.1-5 In the past
several decades, millions of people have benefited from the use of statins in terms of both
extending their lives and improving the quality of life.1 Moreover, extensive researches in the field
indicate that statins have many other pharmacological activities, including anti-inflammatory and
anti-cancer activity, as well as anti-oxidative effects.4, 6-9 Given their pharmaceutical importance,
tremendous efforts have been devoted to the efficient and sustainable synthesis of statins.4, 10-11 In
particular, various methods have been developed to build the chiral syn-3,5-dihydroxy-6-heptenoic
or heptanoic acid side chain, the common and pharmacologically important structural feature of
statins.4 Within this context, our group has longtime interests in the efficient and asymmetric
synthesis of statins.11-16 Recently, we have demonstrated a pilot-plant-scale synthesis of
bromocarbonate 3a via a one-pot diastereoselective carboxylation/bromocyclization of tert-butyl
(R)-3-hydroxyl-5-hexenoate ((R)-2a) (Scheme 1A).17 The bromocarbonate 3a was transformed
further to Kaneka alcohol 4a, the common synthetic intermediate to statin molecules, in three steps
with high yields. In our original synthetic route, enantiomerically pure (R)-2a was produced
through the reduction of ketoester 1a by NaBH4 in the presence of (L)-tartaric acid at -40 ℃.
Although effective, the cryogenic reaction conditions used present limitations. For industrial
application, development of an alternative synthesis of (R)-2a under mild and sustainable
conditions is highly wanted.
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Ketoreductases (KREDs) are a class of enzymes that can reduce prochiral ketones to chiral
alcohols using NADPH or NADH as the hydride donor.18-23 Owing to their excellent
stereoselectivity, broad substrate spectrum, environmental-friendliness, and good stability,
KREDs have been developed as some of most widely used biocatalysts in the industrial synthesis
of pharmaceuticals,24-29 as exemplified in the synthesis of the key intermediates to atorvastatin,24
montelukast,25 and (S)-licarbazepine.26 In fact, KREDs have become the first choice for the
synthesis of chiral alcohols in major pharmaceutical companies nowadays. Herein, we report the
development of a pilot-plant-scale synthesis of (R)-2a through a KRED-catalyzed reduction of 1a
under mild conditions (Scheme 1B).
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Scheme 1. A. New synthetic route to statins recently developed by our group.17 B. KRED-
catalyzed reduction of ketoester 1a to alcohol (R)-2a.
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Screening of ketoreductases. Our study commenced with searching for the most efficient
and selective KRED catalyst. Hence, a small library of in-house preserved KREDs were screened
for the reduction of 1a with the glucose dehydrogenase (GDH)/glucose system employed to
regenerate the cofactor NADPH. As shown in Table 1, most of the enzymes tested reduced 1a to
selectively yield (S)-2a. The desired (R)-2a was only selectively formed in KRED-03-, and KRED-
06-catalyzed reduction reactions (Entry 3 and 6). Fortunately, KRED-06 was exceptionally
effective and able to completely reduce 1a to generate (R)-2a with >99% ee. When the
GDH/glucose system is adopted to regenerate the cofactor NAD(P)H, in particular at high-
substrate loading cases, bases, such as K2CO3 or NaOH have to be routinely added to maintain the
reaction pH because of the formation of gluconic acid.29 To alleviate this inconvenience, we
examined the possibility of using LkADH,30-31 an enzyme known for its ability to regenerate
NADPH via the oxidation of iso-propanol (IPA) to acetone, for the cofactor regeneration. To our
delight, excellent reaction conversion and stereoselectivity were achieved (Entry 12, Table 1).
Therefore, LkADH/IPA was employed for NADPH regeneration in the following studies.
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Table 1. Initial screening of KREDs for the reduction of 1a.a
Entry
1
Enzyme
Conversion (%)b
36.0
Ee (%)c
2.5
Configurationd
R
KRED-01
82.7
>99
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34.0
S
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KRED-02
KRED-03
R
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78.2
>99
>99
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81.6
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>99
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>99
>99
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12e
aReaction conditions (9 mL): 1a (10 g/L), glucose (19.6 g/L), NADP+ (0.0125 g/L), KRED (2.7
g/L), and GDH (1 g/L) in KPi buffer (100 mM, pH 7.0). Reaction mixtures were incubated at 25 ℃
with 200 rpm shaking for 90 min. bThe conversion was determined by GC-MS analysis. cThe ee
was determined by chiral HPLC analysis after benzoylation of the product alcohol. dThe absolute
configuration was assigned by comparing the optical rotation data of the product to that of the
literature data. eLkADH and IPA were used for the regeneration of NADPH.
Effect of substrate concentration. With an effective KRED identified, we next proceeded
to further modify this bioreduction process. Substrate loading was the first parameter we wanted
to optimize. To make a biocatalytic process industrially viable, a substrate concentration of ≥100
g/L is normally required.23 In the present study, a substrate concentration range of 20 g/L to 200
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g/L of ketoester 1a was tested (Figure 1). When 100 g/L or less of 1a was used, >95% conversion
was achieved. When 125 g/L of 1a was subjected to the same reaction conditions, approximately
83% conversion was obtained. Further increase of the substrate loadings to 150 g/L and 200 g/L
resulted in moderate reaction conversions (67% and 49%, respectively). Taking into consideration
of both relatively high substrate loading and good reaction conversion, we chose the substrate
concentration of 125 g/L for the remaining study of process optimization.
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Figure 1. Effect of substrate concentration on the bioreduction of ketone 1a. Reaction conditions
(0.8 mL): 1a (variable concentrations), IPA (2.0 equiv. relative to 1a), NADP+ (0.0125 g/L),
KRED-06 (5 g/L), and LkADH (2.5 g/L) in KPi buffer (100 mM, pH 7.0). Reaction mixtures were
incubated at 25 ℃ with 200 rpm shaking for 12 h. Each data point represents the mean ± S.D.
(range) of triplicate assays.
Effect of IPA equivalent. Efficient regeneration of NADPH plays a pivotal role in KRED-
catalyzed reduction reactions. With this in mind, we next studied the effect of IPA, the sacrificial
substrate used in our catalytic system. As seen in Figure 2, by increasing the amount of IPA from
1 equiv. to 2.5 equiv., the reaction conversion was improved from 75% to 94%. On the other hand,
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using more than 2.5 equiv. of IPA did not improve the reaction conversion further. Hence, 2.5
equiv. of IPA was identified as the optimal amount.
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Figure 2. Effect of concentration of IPA on the reaction conversion. Reaction conditions (0.8 mL):
1a (125 g/L), IPA (variable concentrations), NADP+ (0.0125 g/L), KRED-06 (5 g/L), and LkADH
(2.5 g/L) in KPi buffer (100 mM, pH 7.0). Reaction mixtures were incubated at 25 ℃ with 200
rpm shaking for 12 h. Each data point represents the mean ± S.D. (range) of triplicate assays.
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Effect of reaction temperature and pH. Temperature is another important parameter for
biocatalytic reactions. On one hand, high temperature usually means fast reaction rate, which is
beneficial for an efficient industrial process. On the other hand, unlike chemo-catalysts,
biocatalysts are usually not able to withstand high temperature conditions. Therefore, it is essential
to find out the optimal operating temperature for a given biocatalytic process. Firstly, our study of
temperature on the reaction conversion suggested this bioreduction reaction could occur readily at
the temperature range of 25 to 50 ℃ (Figure 3), with 30 ℃ giving a slightly better conversion.
Secondly, in the temperature-tolerant experiments (Figure 4), our biocatalysts (KRED-06 and
LkADH) retained more than 94% activity after being incubated at 30 ℃ for 24 h. In comparison,
the biocatalysts lost about 22% and 70% activity after being incubated at 35 ℃ and 40 ℃ for 24 h,
respectively. Collectively, 30 ℃ was identified as the optimal reaction temperature.
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Next, the effect of pH on the reaction conversion was examined as well. Apparently, this
biocatalytic reaction functioned well at a pH range of 5.0 to 9.0, with pH 7.0 giving the best results
(Figure 5).
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Figure 3. Effect of temperature on the reaction conversion. Reaction conditions (0.8 mL): 1a (125
g/L), IPA (2.5 equiv. relative to 1a), NADP+ (0.0125 g/L), KRED-06 (5 g/L), and LkADH (2.5
g/L) in KPi buffer (100 mM, pH 7.0). Reaction mixtures were incubated at different temperatures
with 200 rpm shaking for 12 h. Each data point represents the mean ± S.D. (range) of triplicate
assays.
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Figure 4. Activities of KRED-06/LkADH to thermal treatment at 25 ℃, 30 ℃, 35 ℃, and 40 ℃.
The initial activity of KRED-06/LkADH without thermal treatment was defined as 100%. Reaction
conditions: the crude enzyme extracts of KRED-06 and LkADH were incubated at temperatures
of 25 ℃, 30 ℃, 35 ℃, and 40 ℃ for different periods prior to assaying activity.
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Figure 5. Effect of pH on the reaction conversion. Reaction conditions (0.8 mL): 1a (125 g/L),
IPA (2.5 equiv. relative to 1a), NADP+ (0.0125 g/L), KRED-06 (5 g/L), and LkADH (2.5 g/L) in
different buffers, including 100 mM sodium citrate buffer (pH 5.0 or 6.0), 100 mM KPi buffer (pH
7.0 or 8.0), and 100 mM glycine buffer (pH 9.0). Reaction mixtures were incubated at 30 ℃ with
200 rpm shaking for 12 h. Each data point represents the mean ± S.D. (range) of triplicate assays.
Effect of biocatalyst and cofactor loadings. The amounts of biocatalysts and cofactor
used largely determine the production cost of a biocatalytic process. Regarding the former aspect,
when the KRED-06 loading was reduced from 5 g/L to 4 g/L, the reaction was still able to go to
completion (Figure 6). More significantly, our study of the NADP+ loading showed that without
the addition of exogenous NADP+, the bioreduction reaction was able to go to completion,
although a longer reaction time was required compared to those reactions running in the presence
of exogenous NADP+ (Figure 7). These results suggested the endogenous NADP+ present in the
crude enzyme extracts was sufficient to drive the biotransformation to completion, which would
greatly reduce the production cost of this biocatalytic process.
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Figure 6. Effect of biocatalyst loading on the reaction conversion. Reaction conditions (0.8 mL):
1a (125 g/L), IPA (2.5 equiv. relative to 1a), NADP+ (0.0125 g/L), KRED-06 (variable
concentrations), and LkADH (50% w/w relative to KRED-06) in KPi buffer (100 mM, pH 7.0).
Reaction mixtures were incubated at 30 ℃ with 200 rpm shaking for 12 h. Each data point
represents the mean ± S.D. (range) of triplicate assays.
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Figure 7. Effect of cofactor loading on the reaction conversion. Reaction conditions (0.8 mL): 1a
(125 g/L), IPA (2.5 equiv. relative to 1a), NADP+ (variable concentrations), KRED-06 (4 g/L),
and LkADH (2 g/L) in KPi buffer (100 mM, pH 7.0). Reaction mixtures were incubated at 30 ℃
with 200 rpm shaking for 1-17 h.
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Pilot-plant-scale synthesis. After practice at gram- and hundred-gram-scale, the above-
optimized reaction conditions were eventually applied to the synthesis of (R)-2a at a pilot-plant-
scale. Specifically, 1a (3.3 kg, 125 g/L, 90% GC purity), IPA (3.42 L, 2.5 equiv.), crude enzyme
extracts of KRED-06 (4 g/L), and crude enzyme extracts of LkADH (2 g/L) in KPi buffer (23 L,
100 mM, pH 7.0) were stirred in a reactor at 30 ℃. After completion of the reaction as monitored
by GC, the pH was adjusted to 3~4 by the addition of 4 N HCl. Celite was added and the mixture
was stirred for 10 min prior to filtration. The filter cake was extracted with EtOAc for three times,
and this combined organic solution was then used to extract the aqueous filtrate from the first
filtration. The combined EtOAc solution was dried, and concentrated in vacuo to give (R)-2a (3.21
kg, 96.2% yield) with a GC purity of 80%, as well as an enantiomeric purity of >99.9% ee. Without
further purification, thus obtained (R)-2a can be directly used in our reported synthesis of
bromocarbonate 3a via a one-pot diastereoselective carboxylation/bromocyclization sequence.17
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Biocatalytic synthesis of homoallylic alcohols 2. To further show its synthetic potential,
the currently developed biocatalytic system was applied to the synthesis of various homoallylic
alcohols 2 (Scheme 2). Firstly, ketoesters 1c-1g were all efficiently reduced by KRED-06 to give
the desired alcohols in 74%-to-87% isolated yields and excellent enantiomeric purity (all >97%
ee). Ketoester 1b containing an iso-butyl group was reduced in a less stereoselective manner,
yielding the corresponding alcohol (R)-2b with 82% ee. Functional groups, such as chloro group
(1h), cyano group (1i), and ether moieties (1j-1l) were well tolerated under our bioreduction
conditions, with the corresponding homoallylic alcohols 2h-2l being produced in moderate-to-
excellent yields and excellent enantiomeric purity (all >91% ee). Interestingly, the enantiomeric
purity of alcohol (R)-2m bearing a benzyl ether moiety was only 60% ee, much lower than that of
its shorter-carbon-chain counterpart (R)-2j (>99% ee), suggesting the chain length of the substrates
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might affect the binding modes in the KRED-06-catalyzed reduction reactions. Similarly, ketones
with a benzyl group attaching to the carbonyl group (1n-1p) were all reduced by KRED-06 in a
highly stereoselective manner, affording product alcohols with ≥88% ee. In stark contrast,
substrates with an aryl group directly attaching to the carbonyl group (1q and 1r) were reduced by
the enzyme in a completely non-stereoselective fashion, generating racemic products 2q and 2r.
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Scheme 2. KRED-06-catalyzed stereoselective synthesis of enantioenriched homoallylic alcohols
2. aThe absolute configuration was not assigned.
Conclusions
In summary, through screening a small library of in-house preserved KREDs, KRED-06
was identified as an efficient and selective biocatalyst capable of reducing ketoester 1a to generate
tert-butyl (R)-3-hydroxyl-5-hexenoate ((R)-2a). After further reaction optimization, this
biocatalytic process was successfully performed at a pilot-plant-scale without the addition of
exogenous NADP+, producing 3.21 kg of enantiomerically pure (R)-2a (96.2% yield, >99.9% ee).
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Compared to our first-generation synthesis of (R)-2a using NaBH4 and (L)-tartaric acid under
cryogenic conditions (-40 ℃), the currently developed biocatalytic approach features mild reaction
conditions, environmental friendliness, and outstanding stereoselectivity, therefore possessing
great potential to be implemented into industrial production of statins.
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EXPERIMENTAL SECTION
General Methods. Unless otherwise specified, all reagents and solvent were purchased
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from commercial sources and used as received. H (400 MHz) and C (101 MHz) NMR were
recorded on a Bruker Avance 400 spectrometer in CDCl3 using tetramethylsilane (TMS) as internal
standards. Products were purified by flash column chromatography on silica gel purchased from
Qingdao Haiyang Chemical Co., Ltd. Optical rotations were measured by a Rudolph AUTOPOL
I Automatic Polarimeter. HRMS were recorded on a Bruker micrOTOF spectrometer. The
compounds (2a-i, 2k-l, 2p) were benzoylated and their enantiomeric excess was determined by
HPLC with Chiralpak IC column (25 cm × 4.6 mm × 5 μm) and Chiralpak AD-H column (25 cm
× 4.6 mm × 5 μm). The enantiomeric excess of compounds (2j, 2m-o, 2q-r) was determined by
HPLC with Chiralpak OJ column (25 cm × 4.6 mm × 5 μm) and Chiralpak OD-H column (25 cm
× 4.6 mm × 5 μm).
Cloning, Expression (Shake-flask), and crude enzyme extracts (CEE) preparation.
KRED-06 was cloned into the NdeI and BamHI restriction sites of pET-24b. The rest of
ketoreductases in this study were prepared as we previously described.32-34
An approximately 12 h culture of E. coli BL21 (DE3) cells freshly transformed with the
appropriate plasmid and grown in LB medium supplemented with either kanamycin (50 μg/mL)
or ampicillin (100 μg/mL) was diluted 1:100 into 0.5 L of the same medium in a 2 L flask. The
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culture was shaking at 37 °C until the optical density at 600 nm reached 0.6-0.8, then the flask was
placed in an ice/water bath for ca. 30 min before the addition of isopropylthio-β-D-galactoside
(IPTG) to a final concentration of 100 μM. The culture was shaking for an additional 14-16 h at
18 ºC. The cells were collected by centrifugation, and then resuspended in an appropriate amount
of KPi buffer (100 mM, pH 7.0) to make a 15% w/v suspension. The cells were lysed by sonication
on ice and debris was removed by centrifugation at 37,000 x g for 30 min at 4 °C. This collected
supernatant (crude enzyme extracts, CEE) was used as the biocatalyst. The quantification of the
crude enzyme extracts was following the common practice for KREDs.35-38 Briefly, a portion of
the crude enzyme extracts was lyophilized and weighed. Next, this obtained information was used
as the reference for the calculation of the biocatalyst loading (g/L).
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Expression of KRED-06 using 7 L fermenter. An approximately 12 h culture of E. coli
BL21 (DE3) cells freshly transformed with pET-24b-KRED-06 and grown in LB medium
supplemented with kanamycin (50 μg/mL) was inoculated with an inoculation concentration of
1% (v/v) into a 7 L fermenter containing 5 L of a sterilized and modified LB medium (20 g/L
tryptone, 10 g/L Yeast Extract, 10 g/L NaCl, 1.5 g/L glycerol) with kanamycin (100 mg). The
fermentation process was first carried out at 30 °C for approximately 9 h with an aeration of 0.5
vvm (air volume/culture volume/minute). The agitation rate was occasionally adjusted to maintain
the dissolved oxygen concentration above 30%. Then, the temperature was lowered to 25 °C and
the pH was auto-controlled as 6.85 by the addition of ammonia and phosphoric acid. In addition,
glycerol was added at the rate of 10 mL/h. At this point, 100 mg of IPTG was added to induce the
expression. The agitation rate was auto-adjusted to maintain the dissolved oxygen concentration
above 35%. After 14 h of induction, recombinant E. coli cells were harvested as described above,
and stored at −80 °C until further use.
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KRED-06-catalyzed synthesis of (R)-2a at a pilot-plant-scale. The reaction mixture
containing 1a (3.3 kg, 125 g/L, 90% GC purity), IPA (3.42 L, 2.5 equiv.), crude enzyme extracts
of KRED-06 (4 g/L), crude enzyme extracts of LkADH (2 g/L), and KPi buffer (23 L, 100 mM,
pH 7.0) in a 50 L reactor was stirred at 30 ℃. After completion of the reaction as monitored by
GC, the pH was adjusted to 3~4 by the addition of 4 N HCl. Celite was added and the mixture was
stirred for 10 min prior to filtration. The filter cake was extracted with EtOAc for three times and
this combined organic solution was then used to extract the aqueous filtrate from the first filtration.
The combined EtOAc solution was dried, and concentrated in vacuo to give (R)-2a (3.21 kg, 96.2%
yield) with a GC purity of 80%, as well as an enantiomeric purity of >99.9% ee. Without further
purification, thus obtained (R)-2a can be directly used in our reported synthesis of bromocarbonate
3a via a one-pot diastereoselective carboxylation/bromocyclization sequence. A small portion of
this product was subjected to vacuum distillation (58-63 ℃/1-2 mm Hg) to afford (R)-2a for
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1
analytical purpose. Colorless oil; H NMR (400 MHz, CDCl3) δ 5.83-5.69 (m, 1H), 5.06 (d, J =
6.4 Hz, 1H), 5.02 (s, 1H), 3.97 (dd, J = 5.9, 2.9 Hz, 1H), 3.18 (s, 1H), 2.40-2.14 (m, 4H), 1.39 (s,
9H). 13C{1H} NMR (101 MHz, CDCl3) δ 172.2, 134.1, 117.8, 81.1, 67.4, 41.6, 40.9, 28.0.
HRMS(ESI) m/z [M+Na]+ calcd for C10H18O3Na 209.1148, found 209.1144. [α]20D -26.9 (c 1.14,
CHCl3). lit.[α]20 -24.3 (c 1.0, CHCl3).17 HPLC (Chiracel® IC, Hexane : Isopropanol = 90 : 10,
D
Flow rate = 1.0 mL/min, λ = 254 nm, 25 ℃) : t1 = 4.9 min (major enantiomer), t2 = 6.5 min. (>
99.9% ee)
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website.
Experimental details including enzyme information, synthesis of substrates, enzymatic reactions,
product characterization. NMR spectra and chiral-HPLC spectra are provided as well.
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AUTHOR INFORMATION
Corresponding Author
*huangzedu@fudan.edu.cn
*rfchen@fudan.edu.cn
ORCID
Zedu Huang: 0000-0003-3320-3392
Fener Chen: 0000-0002-6734-3388
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial supports from the National Natural Science Foundation of China (no. 21801047) and
Shanghai Sailing Program (18YF1402100) are greatly appreciated.
REFERENCES
(1)
Bifulco, M.; Endo, A. Statin: New Life for an Old Drug. Pharmacol. Res. 2014, 88, 1-2.
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3
4
(2)
Stein, E. A. The Power of Statins: Aggressive Lipid Lowering. Clin. Cardiol. 2003, 26,
5
6
III25-III31.
7
8
9
(3)
(4)
Stossel, T. P. The Discovery of Statins. Cell 2008, 134, 903-905.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Časar, Z. Historic Overview and Recent Advances in the Synthesis of Super-Statins. Curr.
Org. Chem. 2010, 14, 816-845.
(5)
Hoyos, P.; Pace, V.; Alcántara, A. R. Biocatalyzed Synthesis of Statins: A Sustainable
Strategy for the Preparation of Valuable Drugs. Catalysts 2019, 9, 260.
(6)
Liao, J. K. Statins: Potent Vascular Anti-Inflammatory Agents. Int. J. Clin. Pract. 2004,
58, 41-48.
(7)
Sleijfer, S.; van der Gaast, A.; Planting, A. S.; Stoter, G., Verweij, J. The Potential of
Statins as Part of Anti-Cancer Treatment. Eur. J. Cancer 2005, 41, 516-522.
(8)
Stoll, L. L.; McCormick, M. L.; Denning, G. M.; Weintraub, N. L. Antioxidant Effects of
Statins. Drugs Today 2004, 40, 975-990.
(9)
Watanabe, T.; Yasunari, K.; Nakamura, M. Antioxidative Actions of Statins: Potential
Mechanisms for Antiathersclerotic Effects. Mini Rev. Med. Chem. 2006, 6, 505-508.
(10) Miyachi, N.; Suzuki, M.; Ohara, Y.; Hiyama, T. Syntheses of HMG-CoA Reductase
Inhibitors. J. Synth. Org. Chem. Jpn. 1995, 53, 186-196.
(11) Wu, Y.; Xiong, F.; Chen, F. Stereoselective Synthesis of 3-Hydroxy-3-Methylglutaryl-
Coenzyme A Reductase Inhibitors. Tetrahedron 2015, 71, 8487-8510.
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Page 22 of 25
1
2
3
4
5
6
7
8
9
(12) Chen, X.; Xiong, F.; Zheng, C.; Li, J.; Chen, F. Synthetic Studies on Statins. Part 3: A
Facile Synthesis of Rosuvastatin Calcium through Catalytic Enantioselective Allylation Strategy.
Tetrahedron 2014, 70, 5794-5799.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Chen, X.; Xiong, F.; Chen, W.; He, Q.; Chen, F. Asymmetric Synthesis of the HMG-CoA
Reductase Inhibitor Atorvastatin Calcium: An Organocatalytic Anhydride Desymmetrization and
Cyanide-Free Side Chain Elongation Approach. J. Org. Chem. 2014, 79, 2723-2728.
(14) Hu, L.; Xiong, F.; Chen, X.; Chen, W.; He, Q.; Chen, F. Synthetic Studies on Statins. Part
1: A Short and Cyanide-Free Synthesis of Atorvastatin Calcium via an Enantioselective Aldol
Strategy. Tetrahedron-Asymmetry 2013, 24, 207-211.
(15) Wu, Y.; Liu, M.; Huang, H.; Huang, G.; Xiong, F.; Chen, F. Asymmetric Synthesis of
Atorvastatin Calcium through Intramolecular Oxidative Oxygen-Nucleophilic Bromocyclization.
Eur. J. Org. Chem. 2017, 3681-3688.
(16) Xiong, F.; Wang, H.; Yan, L.; Han, S.; Tao, Y.; Wu, Y.; Chen, F. Stereocontrolled
Synthesis of Rosuvastatin Calcium via Iodine Chloride-Induced Intramolecular Cyclization. Eur.
J. Org. Chem. 2017, 3681-3688.
(17) Huang, G.; Liu, M.; Xiong, F.; Meng, G.; Tao, Y.; Wu, Y.; Peng, H.; Chen, F. Chiral Syn-
1,3-diol Derivatives via a One-Pot Diastereoselective Carboxylation/Bromocyclization of
Homoallylic Alcohols. iScience 2018, 9, 513-520.
(18) Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Recent Advances in the Biocatalytic
Reduction of Ketones and Oxidation of Sec-Alcohols. Curr. Opin. Chem. Biol. 2004, 8, 120-126.
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Organic Process Research & Development
1
2
3
4
(19) An, J.; Nie, Y.; Xu, Y. Structural Insights into Alcohol Dehydrogenases Catalyzing
5
6
Asymmetric Reductions. Crit. Rev. Biotechnol. 2019, 39, 366-379.
7
8
9
(20) Chen, B.; de Souza, F. Z. R. Enzymatic Synthesis of Enantiopure Alcohols: Current State
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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41
42
43
44
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47
48
49
50
51
52
53
54
55
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59
60
and Perspectives. RSC Advances 2019, 9, 2102-2115.
(21) Hollmann, F.; Arends, I. W. C. E.; Holtmann, D. Enzymatic Reductions for the Chemist.
Green Chem. 2011, 13, 2285-2314.
(22) Nestl, B. M.; Hammer, S. C.; Nebel, B. A.; Hauer, B. New Generation of Biocatalysts for
Organic Synthesis. Angew. Chem. Int. Ed. 2014, 53, 3070-3095.
(23) Sheldon, R. A.; Brady, D. Broadening the Scope of Biocatalysis in Sustainable Organic
Synthesis. ChemSusChem 2019, 12, 2859-2881.
(24) Ma, S. K.; Gruber, J.; Davis, C.; Newman, L.; Gray, D.; Wang, A.; Grate, J.; Huisman, G.
W.; Sheldon, R. A. A Green-by-Design Biocatalytic Process for Atorvastatin Intermediate. Green
Chem. 2010, 12, 81-86.
(25) Liang, J.; Lalonde, J.; Borup, B.; Mitchell, V.; Mundorff, E.; Trinh, N.; Kochrekar, D. A.;
Cherat, R. N.; Pai, G. G. Development of a Biocatalytic Process as an Alternative to the (-)-DIP-
Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast. Org. Process Res.
Dev. 2010, 14, 193-198.
(26) Modukuru, N. K.; Sukumaran, J.; Collier, S. J.; Chan, A. S.; Gohel, A.; Huisman, G. W.;
Keledjian, R.; Narayanaswamy, K.; Novick, S. J.; Palanivel, S. M.; Smith, D.; Wei, Z.; Wong, B.;
Yeo, W. L.; Entwistle, D. A. Development of a Practical, Biocatalytic Reduction for the
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2
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4
5
6
Manufacture of (S)-Licarbazepine Using an Evolved Ketoreductase. Org. Process Res. Dev. 2014,
18, 810-815.
7
8
9
(27) Guo, X.; Tang, J.; Yang, J.; Ni, G.; Zhang, F.; Chen S. Development of a Practical
Enzymatic Process for Preparation of (S)‑2-Chloro-1-(3,4-difluorophenyl)ethanol. Org. Process
Res. Dev. 2017, 21, 1595-1601.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
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35
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41
42
43
44
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48
49
50
51
52
53
54
55
56
57
58
59
60
(28) Wei, T.; Tang, J.; Ni, G.; Wang, H.; Yi, D.; Zhang, F.; Chen S. Development of an
Enzymatic Process for the Synthesis of (S)‑2-Chloro-1-(2,4-dichlorophenyl) Ethanol. Org.
Process Res. Dev. 2019, 23, 1822-1828.
(29) Gong, X.; Zheng, G.; Liu, Y.; Xu, J. Identification of a Robust Carbonyl Reductase for
Diastereoselectively Building syn-3,5-Dihydroxy Hexanoate: a Bulky Side Chain of Atorvastatin.
Org. Process Res. Dev. 2017, 21, 1349-1354.
(30) Weckbecker, A.; Hummel, W. Cloning, Expression, and Characterization of an (R)-
Specific Alcohol Dehydrogenase from Lactobacillus Kefir. Biocatal. Biotransform. 2006, 24, 380-
389.
(31) Krausser, M.; Hummel, W.; Groger, H. Enantioselective One-pot Two-step Synthesis of
Hydrophobic Allylic Alcohols in Aqueous Medium through the Combination of a Wittig Reaction
and an Enzymatic Ketone Reduction. Eur. J. Org. Chem. 2007, 5175-5179.
(32) Li, Z.; Wang, Z.; Wang, Y.; Wu, X.; Lu, H.; Huang, Z.; Chen, F. Substituent Position-
Controlled Stereoselectivity in Enzymatic Reduction of Diaryl- and Aryl(heteroaryl)methanones.
Adv. Synth. Catal. 2019, 361, 1859-1865.
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(33) Wang, Z.; Wu, X.; Li, Z.; Huang, Z.; Chen, F. Ketoreductase Catalyzed Stereoselective
5
6
Bioreduction of α-Nitro Ketones. Org. Biomol. Chem. 2019, 17, 3575-3580.
7
8
9
(34) Wang, Z.; Zeng, Y.; Wu, X.; Li, Z.; Tao, Y.; Yu, X.; Huang, Z.; Chen, F. Access to Chiral
α-Substituted-β-Hydroxy Arylphosphonates Enabled by Biocatalytic Dynamic Reductive Kinetic
Resolution. Org. Biomol. Chem. 2020, 18, 2672-2677.
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(35) Li, C.; Liu, Y.; Pei, X.; Wu, Z. Stereo-complementary Bioreduction of Saturated N-
Heterocyclic Ketones. Process Biochem. 2017, 56, 90-97.
(36) Huisman, G. W.; Liang, J.; Krebber, A. Practical Chiral Alcohol Manufacture Using
Ketoreductases. Curr. Opin. Chem. Biol. 2010, 14, 122-129.
(37) Kosjek, B.; Nti-Gyabaah, J.; Telari, K.; Dunne, L.; Moore, J. C. Preparative Asymmetric
Synthesis of 4,4-Dimethoxytetrahydro-2H-pyran-3-ol with a Ketone Reductase and in situ
Cofactor Recycling Using Glucose Dehydrogenase. Org. Process Res. Dev. 2008, 12, 584-588.
(38) Gooding, O, W.; Voladri, R.; Bautista, A.; Hopkins, T. Development of a Practical
Biocatalytic Process for (R)-2-Methylpentanol. Org. Process Res. Dev. 2010, 14, 119-126.
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