Appl Microbiol Biotechnol (2014) 98:11–21
DOI 10.1007/s00253-013-5357-0
MINI-REVIEW
Chemical and enzymatic approaches to the synthesis
of optically pure ethyl (R )-4-cyano-3-hydroxybutanoate
Zhong-Yu You & Zhi-Qiang Liu & Yu-Guo Zheng
Received: 14 September 2013 / Revised: 21 October 2013 / Accepted: 22 October 2013 / Published online: 15 November 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Ethyl (R )-4-cyano-3-hydroxybutanoate (HN) is an
important chiral synthon for side chain of the cholesterollowering drug atorvastatin (Lipitor), which is the
hydroxymethylglutaryl CoA reductase inhibitor. HN is also
used as a synthon in the production of L -carnitine and (R )-4amino-3-hydroxybutanoic acid. It is necessary to have a clear
understanding of the synthesis process of HN for its extensive
use. This review gives an overview of different synthetic
strategies of optically active HN, including chemical and
enzymatic approaches. The emphasis is focused mainly on
the synthetic routes using biocatalysts, such as halohydrin
dehalogenase, nitrilase, carbonyl reductase, and lipase.
Keywords Ethyl (R)-4-cyano-3-hydroxybutanoate .
Syntheticapproach . Chemicalsynthesis . Enzymaticsynthesis
Introduction
The majority of drugs are constituted by chiral molecules, and
the two enantiomers may have significant differences in biological activities such as pharmacology, toxicology, pharmacokinetics, metabolism, etc. (Brooks et al. 2011; Hutt and
O’Grady 1996; Nguyena et al. 2006). Therefore, chiral compounds have gained increasing attention in the pharmaceutical
field (Patel 2006; Patel 2008). They are useful starting materials in the synthesis of drug substances. Chiral 4-substituted
3-hydroxybutyric acid derivatives, such as ethyl (S)-4-chloro3-hydroxybutanoate ((S )-CHBE), ethyl (S )-4-bromo-3-
hydroxybutyrate ((S )-BHBE), and ethyl (R )-4-cyano-3hydroxybutanoate (HN), are commercially important building
block in the pharmaceutical production (Davis et al. 2004,
2006; Patel 2009). HN is mainly used for the production of
atorvastatin (Lipitor), which is a synthetic cholesterollowering agent (Fig. 1) (Brower et al. 1992; Roth 1987; Tao
and Xu 2009). Atorvastatin selectively inhibits 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase that catalyzes the conversion of HMG-CoA to mevalonate in the early
and rate-limiting step of cholesterol biosynthesis (BarriosGonzalez and Miranda 2010; Roth 2002). HN is also a
synthon in the production of L -carnitine and (R)-4-amino-3hydroxybutanoic acid (GABOB) (Fig. 1) (Wang and
Hollingsworth 1999). L -Carnitine is known as a very important quaternary ammonium compound in β-oxidation of fatty
acids in mammals (Jung et al. 1993; Wang and Hollingsworth
1999), and GABOB is an anticonvulsant drug (Candela et al.
2000; Mete et al. 2003).
Because of its important use in the pharmaceutical industry,
several different synthetic approaches have been developed
for the synthesis of HN. The synthesis of single enantiomer
can be executed by chemical or biocatalytic methods
(Pantaleone 2005). Although a number of literatures have
been published on the synthesis of (S )-CHBE (Kataoka
et al. 2003; Kita et al. 1999; Ye et al. 2011) and (S)-BHBE
(Asako et al. 2009), the review specifically focused on the HN
synthesis that have not been reported so far. This paper gives
information on the production of HN by various routes, and
the advantages and disadvantages of each synthesis route are
discussed.
Z.<Y. You : Z.<Q. Liu : Y.<G. Zheng (*)
Institute of Bioengineering, Zhejiang University of Technology,
Hangzhou, Zhejiang 310014, People’s Republic of China
e-mail: [email protected]
Chemical synthesis of ethyl
(R )-4-cyano-3-hydroxybutanoate
Z.<Y. You : Z.<Q. Liu : Y.<G. Zheng
Engineering Research Center of Bioconversion and Biopurification
of the Ministry of Education, Zhejiang University of Technology,
Hangzhou, Zhejiang 310014, People’s Republic of China
Chemical synthesis is a useful method for construction of complex chemical compounds from simple ones. By chemical synthesis, many substances important to daily life are obtained, such
12
Appl Microbiol Biotechnol (2014) 98:11–21
Fig. 1 Pharmaceuticals derived
from ethyl (R)-4-cyano-3hydroxybutanoate
as shikimic acid (Jiang and Singh 1998), proanthocyanidins (He
et al. 2008b), heterocyclic-sugar nucleoside analogues (Romeo
et al. 2010), etc. To our knowledge, the first report on the
synthesis of ethyl 4-cyano-3-hydroxybutanoate can be traced
back to 1923 (Lespieau 1923). The product was obtained after
hydrolysis, esterification, and cyanidation by using 4-chloro-3hydroxybutyronitrile as substrate. However, the optical activity
was not mentioned and the yield was low. Recently, many
synthetic methods for R-enantiomer using different substrates
as starting material have been developed (Kumar et al. 2005; Roh
et al. 2003).
acid. (S)-CHBE was protected with hexamethyl disilazane,
and further subjected to cyanidation. HN was obtained after
deprotection with hydrochloric acid in the total yield of 57 %.
The specific rotation of the product was calculated ([α]25
D =
−32.4°, c 1.0, CHCl3), which is similar to that in previous
report (Kumar et al. 2005). This route has been applied for HN
production in Zhejiang Neo-Dankong Pharmaceutical Co.
The raw material, (S)-ECH, can be prepared from the epoxide
hydrolase-catalyzed kinetic resolutions of cheap racemic epichlorohydrin (Liu et al. 2011c). If the yield of HN can be
improved, this conversion process has the broad application
prospects in HN production.
Synthesis of HN from (S)-epichlorohydrin
Synthesis of HN from L -malic acid
(S)-epichlorohydrin ((S)-ECH) is a valuable epoxide intermediate in organic synthesis (Liu et al. 2011c). It is very common
to start with (S)-ECH for the preparation of optically active
pharmaceuticals, such as Linezolid (Rajesh et al. 2011), (S)timolol (Narina and Sudalai 2007), and (+)-yatakemycin
(Okano et al. 2008). Hong and Jiang have reported a novel
process for the preparation of HN from (S)-ECH, as shown in
Fig. 2 (Hong and Jiang 2009; Jiang and Hong 2012). (S)-ECH
was ring-opened with sodium cyanide to form (S)-4-chloro-3hydroxybutyronitrile, which was then hydrolyzed and esterified to (S)-CHBE in the presence of alcohol and hydrochloric
Fig. 2 Synthesis of HN from (S)epichlorohydrin
Recently, an alternative process based on L -malic acid for the
HN production has been reported (Lv et al. 2009). L -Malic
acid was converted to (S )-malic acid diethyl ester via esterification, then asymmetric reduction with borane to form (3S )-3,
4-dihydroxybutyric acid ethyl ester (Fig. 3). Subsequently,
(3S)-3,4-dihydroxybutyric acid ethyl ester was bromized with
hydrogen bromide, and cyanided with sodium cyanide in
EtOH/H2O to give HN. As a result, the overall yield of the
process is about 56.7 %. [α ]25
D =−31.02° (c 1.0, CHCl3).
Though L -malic acid is an inexpensive and easily obtained
Appl Microbiol Biotechnol (2014) 98:11–21
13
Fig. 3 Synthesis of HN from L malic acid
starting material, there are two significant drawbacks in the L malic acid process that limit its industrial application: one is
that the yield on the overall process is low; another is that,
borane, the reducing agent in this process, is rather expensive
and extremely dangerous (Guercio et al. 2009). The reduction
reaction should be carried out under the protection of nitrogen,
which increased production costs.
Synthesis of HN from maltodextrin
In 1999, Wang and Hollingsworth reported a process for HN
production (Wang and Hollingsworth 1999). In that case, HN
was prepared via bromization and cyanation processes by
using (S)-3-hydroxy-γ-butyrolactone ((S)-HGB) as substrate.
The yield was 84.5 %; [α]25
D =−31.3° (c 1.0, CHCl3). In later
study, Kumar et al. developed a similar process by using
maltodextrin as starting material to synthesize HN (Kumar
et al. 2005). Reaction of maltodextrin with sodium hydroxide
and cumene hydroperoxide provided 3,4-dihydroxybutyric
acid (Fig. 4). Cyclization of the hydroxy acid, followed by
addition of concentrated sulfuric acid, afforded (S)-HGB. (S)HGB was transformed to HN by bromization and cyanation
processes with the yield of 85 and 50 %, respectively. The
specific rotation of HN was −32.5° (c 1.0, CHCl3). One of
advantages of this process is that this method is simple,
practical, and economical. There are no expensive and unstable reagents involved in the conversion reaction.
Synthesis of HN from (S)-3,4-epoxybutyric acid salt
There is another route to synthesis of HN by chemical method
(Roh et al. 2003), using (S)-3,4-epoxybutyric acid salt as a
Fig. 4 Synthesis of HN from
maltodextrin
starting material, which included cyanation and esterification
(Fig. 5). (S)-3,4-Epoxybutyric acid salt, synthesized from (S)3-hydroxy-γ-butyrolactone, was cyanided by 30 % of aqueous sodium cyanide solution. Then, the reaction mixture was
acidified to pH 1 with concentrated sulfuric acid. Subsequently, the acidic solution was condensed, dissolved in ethanol,
and filtered. The filtrate was refluxed for 5 h and neutralized
with sodium carbonate. Then, the HN with the yield of over
90 % and optical purity of 99.8 % was obtained. The yield in
this route is the highest one in chemical strategies in
literatures.
Cyano-introducing reaction in the HN producing
In summary, chemical synthesis of HN is mainly based on a
cyano-introducing reaction using an ethyl (S )-4-halo-3hydroxybutanoate ((S )-HHBE) as substrate, such as (S )CHBE (Hong and Jiang 2009) and (S )-BHBE (Kumar et al.
2005; Lv et al. 2009; Wang and Hollingsworth 1999), which is
shown in Fig. 6. The difference between these routes is that
(S)-HHBE was derived from various compounds. In addition
to the synthesis methods discussed above, (S)-HHBE also can
be easily synthesized by asymmetric reduction of ethyl 4-halo3-oxobutanoate which was prepared via the diketene route
using either chlorine or bromine (Fig. 7) (Hoff and Anthonsen
1999; Sundby et al. 2003).
Mitsuhashi et al. described a process for synthesis of HN
from (S)-CHBE by a cyano-introducing reaction (Mitsuhashi
et al. 1995). The cyano-introducing reaction happened in an
aprotic polar solvent including dimethyl sulfoxide,
dimethylformamide, water, ethanol, acetonitrile, and tetrahydrofuran; among them water is regarded as the most ideal
14
Appl Microbiol Biotechnol (2014) 98:11–21
Fig. 5 Synthesis of HN from (S)3,4-epoxybutyric acid salt
solvent because it is cheap and it renders possible extraction
with organic solvent (Matsuda et al. 1999).
It should be noted that the yield of this cyano-introducing
reaction is greatly affected by the temperature. The yield was
only 50 % when the reaction was performed at room temperature overnight (Kumar et al. 2005). When the reaction temperature was increased to 50 °C, the yield was raised to 84.5 %
and the reaction time was decreased to 3 h (Wang and
Hollingsworth 1999). As the temperature continued to raise,
it would take over 2 h to complete the reaction, but the yield
was decreased to 57.5 % (Mitsuhashi et al. 1995). These
phenomena can be explained by the effects of temperature
on the reaction rate and stability of HN. At room temperature,
the reaction does not proceed at a reasonable rate because of
the high activation energy. With increasing temperature, the
substance molecules move faster, and the reaction rate is also
increased greatly. Therefore, raising the reaction temperature
is favorable to enhance the yield and shorten the reaction time.
However, the yield was declined when the temperature was
too high. It might be caused by the decomposition of HN
because the product was unstable in polar solvent in thermal
environment. We have studied the stability of aqueous solution of HN at different temperatures (Fig. 8) (not published
data). The result showed that HN was more stable when the
temperature was lower than 45 °C. The retention rate of HN
was 92.7 % after incubation at 45 °C for 8 h, while that was
37.5 % when the temperature was increased to 60 °C. Therefore, the reaction rate and stability of HN must be balanced in
the application of the process. The temperature must be maintained at appropriate point to keep a fast rate of reaction as
well as to keep HN stable.
The main disadvantage of this cyano-introducing reaction is
the generation of by-products, including hydroxyacrylate, cyanoacrylate, 3-cyanobutyrolactone, 3-hydroxybutyrolactone, γcrotonolactone, etc. (Matsuda et al. 1999). These compounds
increased the difficulty of separation and purification of HN. To
overcome this difficulty, a distillation process at 10 Torr with odichlorobenzene as solvent was constructed (Matsuda et al.
Fig. 6 Cyano-introducing
reaction
1999). The HN was purified to a concentration of 94.2 % by
weight with a distillation yield of 96 % by this process.
Enzymatic synthesis of ethyl
(R )-4-cyano-3-hydroxybutanoate
Chemical synthesis is a key tool for making useful substances,
and made significant contributions to our lives. However, the
chemical synthesis in industrial production also brings us a
series of problems, especially the pollution of the environment
(Garrett 1996). Therefore, green chemistry with environmentally friendly, clean, and atom economy features has received
considerable attention in recent years (Anastas and Eghbali
2010). With the development of biotechnology, particularly in
areas such as protein engineering (Bottcher and Bornscheuer
2010; Turner 2009), biocatalysis has fulfilled many key
criteria of green chemistry which has 12 principles (Anastas
and Eghbali 2010). Most of the chemical reactions can be
catalyzed by enzymes (Findrik and Vasic-Racki 2009; Humble and Berglund 2011). The following sections are intended
to provide a general knowledge about the applications of
biocatalyst in the HN synthesis.
The applications of halohydrin dehalogenase in HN synthesis
The biocatalytic dehalogenation is a good example of a biocatalytic reaction. The enzyme involved in dehalogenation is
halohydrin dehalogenase (HHDH, EC 4.5.X.X), which can
degrade a halohydrin to its corresponding epoxide without the
need of any cofactors for keeping its activity (Archelas and
Furstoss 1997; van den Wijngaard et al. 1991; You et al.
2013b). HHDH also can catalyze the reverse reaction (epoxide
ring opening) in the presence of nucleophiles such as
cyanide-, azide-, and nitrite ions (Majerić Elenkov et al.
2006). Therefore, HHDH could be a potential candidate for
the cyano-introducing reaction mentioned above in HN production (Fox et al. 2007; Majeric Elenkov et al. 2006). The
Appl Microbiol Biotechnol (2014) 98:11–21
15
Fig. 7 Asymmetric reduction of
ethyl 4-halo-3-oxobutanoate
used to asymmetric reduction of ethyl-4-chloroacetoacetate by
using a NADP-dependent glucose dehydrogenase for cofactor
regeneration. With 160 g/L of substrate and 0.9 g/L
biocatalysts, the reaction was completed in 8 h and provided
(S)-CHBE in 96 % recovered yield and >99.5 % e.e. In the
second step, the HHDH was employed to catalyze the cyanointroducing reaction at neutral pH and ambient temperature.
With 140 g/L of (S )-CHBE and 1.2 g/L biocatalysts, the
reaction was completed in 5 h and provided HN in 92 %
recovered yield and >99.5 % e.e. This new process has been
scaled-up to 2,000 L reactors, indicated that it is a feasible
route to synthesis of HN.
The applications of nitrilase in HN synthesis
Among the hydrolase family, nitrilase (EC 3.5.5.1) is one of
the most important tools in organic synthesis, which is found
in bacteria, fungi, and plants (Chen et al. 2009). Nitrilase can
convert nitrile compounds to the corresponding carboxylic
acid (Liu et al. 2011a, b). And they have a broad substrate
range containing aliphatic nitriles, aromatic nitriles, and heterocyclic nitriles (Chen et al. 2008; Jin et al. 2011; Liu et al.
2011b; Shen et al. 2009; Xue et al. 2011). Due to its
enantioselectivity, nitrilase has been employed in the synthesis
of chiral compounds such as (R )-(−)-mandelic acid (Kaul
et al. 2004), (S)-(+)-ibuprofen (Yamamoto et al. 1990), and
(S)-naproxen (Kakeya et al. 1991). Another feature of nitrilase
is regioselectivity, which leads to the asymmetric reduction of
100
3h
8h
80
Retention rate (%)
catalytic process under HHDH can be divided into ringclosure and ring-opening. (S )-CHBE was firstly converted
into ethyl (S)-3,4-epoxybutanoate by liberating of a chloride
ion. Then, ethyl (S)-3,4-epoxybutanoate was converted into
HN in the presence of nucleophile (CN−) (Fig. 9).
HHDH from Agrobacterium radiobacter has the most
potential to catalyze the transformation between (S)-CHBE
and HN (Janssen et al. 2006), which has been widely studied,
and its nucleotide sequence, X-ray structure, and catalytic
mechanism have been previously solved (de Jong et al.
2003, 2005; Tang et al. 2003; van Hylckama Vlieg et al.
2001). Unfortunately, the wild-type enzymes showed low
activity and poor stability (Tang et al. 2002, 2005). It catalyzed
the cyano-introducing reaction with a volumetric productivity
of 0.006 g product per liter per hour per gram of catalyst,
which was far from the industrial application demands (Fox
et al. 2007). In the drive for enhanced synthetic efficiency, a
multivariate protein optimization strategy based on protein
sequence activity relationships was used to improve the catalytic properties of HHDH (Fox et al. 2007). A mutant enzyme
with great advantage was obtained after 18 rounds of evolution. The volumetric productivity based on the mutant enzyme
was increased ∼4,000-fold. The comparison of tertiary structure between mutant and wild-type enzymes by homology
modeling showed that the substrate binding pocket was more
open after mutagenesis (Fig. 10a, b), due to which (S)-CHBE
could enter the active site easier. Moreover, the molecular
docking results showed that the positions of (S)-CHBE in
the two active sites are different (Fig. 10c, d). In the mutant
HHDH, the distance from the hydroxyl of (S )-CHBE to
Tyr145, which is the catalytic residue of the enzyme, is much
closer. Therefore, Tyr145 is easier to abstract proton from the
hydroxyl group. In addition, the wild-type enzyme was found
to be susceptible to inactivation under oxidizing conditions
(Tang et al. 2002). The main factor in this inactivation process
is the oxidation of cysteine residues. By replacement of cysteine residues, two more stable mutant enzymes (C153S and
C30A) were obtained which showed a similar activity as wild
type (Tang et al. 2002). Recently, another two engineered
variants, HheC2360 and HheC2656, with improved catalytic
rates and temperature stability were obtained and characterized (Schallmey et al. 2013).
Ma et al. from Codexis had constructed an efficient, scalable, and enzyme-catalyzed process for synthesis of HN utilizing three enzymes including ketoreductase, glucose dehydrogenase, and HHDH (Ma et al. 2010). This process was
divided into two steps. In the first step, a ketoreductase was
60
40
20
0
30
35
40
45
50
60
o
Temperature ( C)
Fig. 8 Thermal stability of ethyl (R)-4-cyano-3-hydroxybutanoate. The
aqueous solution of HN (50 g/L) was incubated at different temperature
for varying periods of time. The retention rate of HN was calculated using
the following formula: Retention rate=C a /C i ×100 %, where C a is the
concentration of HN after incubation and C i is the initial concentration of
HN
16
Appl Microbiol Biotechnol (2014) 98:11–21
Fig. 9 Synthesis of HN by
halohydrin dehalogenase
dinitriles. For example, the aliphatic nitrilase from Acidovorax
facilis 72W could convert 2-methylglutaronitrile to 4cyanopentanoic acid (Chauhan et al. 2003). Therefore, when
the prochiral dinitriles is used as a substrate, one chiral center
can be generated by the nitrilase-catalyzed asymmetric reduction. This process has been used to synthesize HN from 3hydroxyglutaronitrile (Fig. 11).
Robertson et al. discovered and characterized 137 unique
nitrilases from environmental DNA libraries (Robertson et al.
2004). Among them, one (R )-specific nitrilase can catalyze
the desymmetrization of 3-hydroxyglutaronitrile to provide
(R )-4-cyano-3-hydroxybutyric acid on the gram scale. After
reaction, HN was isolated in 98 % yield and 95 % e.e.
(DeSantis et al. 2002). However, the enantiomeric excess
was declined when the concentration of substrate was increased, which limited its large-scale application. Hence,
DeSantis et al. (2003) employed a novel directed-evolution
technique, the gene site saturation mutagenesis method, to
improve the regioselectivity of nitrilase. Finally, they obtained
the best mutant from 31,584 clones via a novel highthroughput screening by using the chiral 15 N-(R )-3hydroxyglutaronitrile as substrate. With 3 M concentration
of substrate, the bioconversion was completed in 15 h and
afforded (R )-4-cyano-3-hydroxybutyric acid in 96 % isolated
(a)
(c)
(b)
(d)
Fig. 10 Surface representation of a wild-type HHDH and b mutant
HHDH. The substrate binding pockets are indicated by arrows. Docking
of (S)-CHBE into the active site of HHDH. c Wild type; d mutant. The
structure of wild-type HHDH was obtained from PDB database (PDB
code: 1PWX). The structure of mutant HHDH was generated by Build
Homology Models (MODELER) in Discovery Studio (DS) 2.1
(Accelrys) using the structure of wild HHDH as a template. Molecular
docking was also performed using the Dock Ligands Module (LibDock)
in DS 2.1. All protein structure figures were prepared with PyMOL
(www.pymol.org)
Appl Microbiol Biotechnol (2014) 98:11–21
17
Fig. 11 Synthesis of HN by
nitrilase
yield and 98.5 % e.e. The volumetric productivity of the
process is 619 g per liter per day.
Nevertheless, the disadvantage of this process is that the
substrate 3-hydroxyglutaronitrile is difficult to synthesis in
large-scale. Bergeron et al. have created a three-step process of HN production by using low-cost epichlorohydrin
as starting material (Bergeron et al. 2006). The epichlorohydrin was cyanided by sodium cyanide to provide 3hydroxyglutaronitrile. Then, the nitrilase-catalyzed biotransformation was performed at optimum conditions:
3 M concentration of 3-hydroxyglutaronitrile, 6 wt% of
enzyme loading, pH 7.5, and 27 °C. After 16 h reaction,
(R )-4-cyano-3-hydroxybutyric acid with 100 % conversion
and 99 % e.e. was obtained. At last, the (R )-4-cyano-3hydroxybutyric acid was esterified with ethanol, and crude
HN was produced in 99 % yield and 98.7 % e.e. A similar
catalytic process has been applied for a patent (Burk et al.
2011).
The applications of carbonyl reductase in HN synthesis
Carbonyl reductases (EC 1.1.1.184) belong to the short-chain
dehydrogenases/reductases protein superfamily (Hoffmann
and Maser 2007). They are widespread in nature and catalyze
the reduction of many carbonyl compounds. The asymmetric
reduction of prochiral ketones with carbonyl reductase is an
effective approach that widely used in the synthesis of
synthons for pharmaceuticals and fine chemicals (Zhu et al.
2005). In a typical application, (S)-CHBE was synthesized by
carbonyl reductase using ethyl 4-chloro-3-oxobutanoate
(COBE) as substrate (Kizaki et al. 2001; You et al. 2013a).
A NADH-dependent carbonyl reductase from Streptomyces
coelicolor has been cloned and expressed in Escherichia coli,
and converted 600 g/L of COBE, which is the highest concentration of substrate in literatures, to (S)-CHBE in a yield of
93 % and an enantioselectivity of >99 % e.e. (Wang et al.
2011). Inspired by this formation process, scientists imagined
a way to apply this to the production of HN, which is direct
synthesis of HN from its β-ketoester precursor (Fig. 12).
He’s group recently reported the synthesis of HN
from ethyl 4-cyano-3-oxobutanoate as starting material
(He et al. 2008a). The whole cells of Bacillus pumilus
Phe-C3 were used as biocatalyst. The substrate was
consumed in a conversion of 100 % after 24 h reaction,
and the product was obtained with 83.1 % isolated yield
and 97 % e.e. The reuse batches of the cells of B.
pumilus Phe-C3 were also investigated. The cells
retained more than 80 % of the initial activity after
six reaction cycles, indicated that the cells were relatively stable under the reaction conditions. Whole-cell
reduction system has the advantages over the enzyme
purification and cofactor regeneration. However, the
substrate concentration was too low to meet the requirements for effective industrial processing. Though they
later optimized the conditions for cell growth and biotransformation, cells tolerated the highest concentration
of ethyl 4-cyano-3-oxobutanoate which still only
20 mM (Jin and Zhang 2012). Wakita has also patented
the same bioconversion process, where several recombinant carbonyl reductases were used (Wakita 2004). In
this process, the concentration of substrate was increased to 135 mM. Ethyl 4-bromo-3-oxobutanoate
was used as raw material to synthesize ethyl 4-cyano3-oxobutanoate with a yield of 56.8 %, which limited
the applications of this process.
However, coenzyme regeneration in the carbonyl reduction
is another key factor to consider. Because of the high price of
coenzyme, the addition of a large amount of coenzyme is
impossible in industrial scale. The effectiveness of wholecell reduction system is very low. So, it is necessary to create
an efficient and economical coenzyme regeneration system
for the carbonyl reduction process (Leonida 2001). Generally,
a coenzyme regeneration system was consisted of one enzyme
and two substrates or two enzymes and two substrates. For
example, the alcohol dehydrogenase from Leifsonia can produce (R)-form chiral alcohols from ketones using 2-propanol
as an auxiliary substrate for the regeneration of cofactor
(Inoue et al. 2006). The carbonyl reductase from Candida
magnoliae and the glucose dehydrogenase from Bacillus
megaterium were used to catalyze the asymmetric reduction
of COBE to (S)-CHBE (Kizaki et al. 2001). In this process,
glucose was used as a hydrogen donor for the cofactor regeneration. In addition, lactate dehydrogenase, glutamate dehydrogenase, and NADH oxidase were also used in the cofactor
Fig. 12 Synthesis of HN by carbonyl reductase
18
Appl Microbiol Biotechnol (2014) 98:11–21
Fig. 13 Synthesis of HN by lipase
regeneration system (Lunzer et al. 1998; Presecki and VasicRacki 2009).
reaction gave 98 % e.e. and 95 % yield. The products were
converted into HN using chlorosulfonylisocyanate or 1-(3dimethylaminopropyl)-3-ethylcarbodiimide (Moen et al.
The applications of lipase in HN synthesis
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is an enzyme that catalyzes the hydrolysis, ammonolysis, and esterification reactions between esters and corresponding acids and
alcohols. It has been widely used in various industrial applications including detergent, food, pharmaceutical, agrochemical, textile, and paper industries (Hasan et al. 2006; Houde
et al. 2004; Liu et al. 2012a). Chiral resolution is an important
tool in the production of optically active compounds from
their racemic compounds. Based on the stereoselectivity, lipase has been successfully used as a catalyst for the chiral
resolution of racemic compounds (Bhushan et al. 2011). Racemic ethyl 4-cyano-3-hydroxybutyrate can be separated into
enantiomers by stereospecific hydrolysis using lipase (Fig. 13
route A). The docking analysis of crystal structure of Candida
antarctica lipase B (PDB code: 1TCA) (Uppenberg et al.
1994), (S)- and (R )-ethyl 4-cyano-3-hydroxybutyrate in the
active site showed that the distance between Ser105 and
carbonyl of ethyl (S)-4-cyano-3-hydroxybutyrate was 2.5 Å,
and the carbonyl of ethyl (R )-4-cyano-3-hydroxybutyrate was
far from Ser105 (Fig. 14). According to the catalytic mechanism of lipase-catalyzed ester hydrolysis (Bornscheuer and
Kazlauskas 1999), the nucleophilic attack of the serine residue
on the carbonyl was the first step which cannot hydrolyze
ethyl (R )-4-cyano-3-hydroxybutyrate.
Recently, Hwang and Chung (2009) has constructed a
process for preparing optically active β-hydroxybutyl ester
by stereospecific hydrolysis of racemic β-hydroxybutyl ester
using lipase or lipase-producing microorganisms. Racemic
ethyl 4-cyano-3-hydroxybutyrate was used as substrate and
the commercial lipase (Novozyme 435) was used as a biocatalyst. HN was obtained with 99 % e.e. after reaction for 3 h.
One disadvantage of this process is that only 50 % of a desired
enantiomer is obtained.
There is another way to produce HN using lipase as biocatalyst, in which the prochiral diethyl 3-hydroxyglutarate was
used as substrate and asymmetric hydrolyzed or ammonolyzed
by lipase (Novozyme 435) (Fig. 13 route B). The ammonolysis
(a)
(b)
Fig. 14 Docking of (S)- and (R)-ethyl 4-cyano-3-hydroxybutyrate into
the active site of CALB. The structure of CALB was obtained from PDB
database (PDB code: 1TCA). Molecular docking was performed using
the Dock Ligands Module (LibDock) in DS 2.1. All protein structure
figures were prepared with PyMOL (www.pymol.org)
Appl Microbiol Biotechnol (2014) 98:11–21
2004). However, the high cost of catalysts in the last step
limited the application of this method.
Concluding remarks
Ethyl (R)-4-cyano-3-hydroxybutanoate is an important building block in the pharmaceutical production. This review concentrated on the methods for the synthesis of HN, including
chemical synthesis and enzymatic synthesis, and attempted to
outline an economical, environmentally friendly, scalable,
reliable, efficient process of HN production. Currently, HN
is mainly produced by the chemical synthesis. The cyanointroducing reaction in chemical synthesis is a simple, practical, and economical process. The highest yield of HN in such
reaction was over 90 %. But the separation and purification of
HN from variety of by-products is very difficult, which
prompted researchers to find a more appropriate method.
Enzymatic synthesis is considered as a useful alternative
compared with traditional chemical ways. Halohydrin
dehalogenase, nitrilase, carbonyl reductase, and lipase are
involved in the enzymatic synthesis of HN. The key point of
enzymatic synthesis is a high catalytic activity of these enzymes at high substrate concentrations, broad pH, and temperature variations. With the development of genetic engineering and protein engineering technologies, such as direct evolution, the enzyme performances have been greatly improved.
Using of these technologies, the halohydrin dehalogenasecatalyzed process and nitrilase-catalyzed process have
achieved in large scale. These synthetic approaches have
provided valuable lessons not only for the production of HN
but also for the synthesis of numerous structurally related
chemicals.
Acknowledgments The authors gratefully acknowledge the financial
supports of the National High Technology Research and Development
Program of China (863 Program) (No. 2011AA02A210075), the Major
Basic Research Development Program of China (973 Project)
(2011CB710804), and Natural Science Foundation of Zhejiang Province
(No. Z4090612, Y4110409 and No. R3110155).
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