Lipase-catalyzed hydrolysis of 2-(4-hydroxyphenyl)propionic acid ethyl ester to (R)-(?)-2-(4-hydroxyphenyl)propanoic acid

Article abstract of DOI:10.1007/s11696-019-00796-9

Stereoselective hydrolysis of (±)-2-(4-hydroxyphenyl)propionic acid ethyl ester (2-HPPAEE) by lipase catalyzed in aqueous system was investigated. Lipase AK with higher catalytic activity and enantioselectivity was selected as catalyst. Simultaneously, factors affecting the conversion of substrate (c) and the enantiomeric excess of product (ee_p) were optimized. The optimal conditions were established, involving 45?°C of temperature, 5.5 of pH, 10?mg of lipase AK dosage, 0.04?mmol of substrate dosage and 40?h of reaction time. Under the optimum conditions, c and ee_p could reach up to 49% and 98%, respectively.

Full text of DOI:10.1007/s11696-019-00796-9

Chemical Papers
https://doi.org/10.1007/s11696-019-00796-9
ORIGINAL PAPER
Lipase‑catalyzed hydrolysis of 2‑(4‑hydroxyphenyl)propionic acid
ethyl ester to (R)‑(−)‑2‑(4‑hydroxyphenyl)propanoic acid
Xin Yuan1 · Panliang Zhang1 · Guangyong Liu1 · Weifeng Xu1 · Kewen Tang1
Received: 9 January 2019 / Accepted: 22 April 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
Stereoselective hydrolysis of ( )-2-(4-hydroxyphenyl)propionic acid ethyl ester (2-HPPAEE) by lipase catalyzed in aqueous
system was investigated. Lipase AK with higher catalytic activity and enantioselectivity was selected as catalyst. Simulta-
neously, factors afecting the conversion of substrate (c) and the enantiomeric excess of product (eep) were optimized. The
optimal conditions were established, involving 45 °C of temperature, 5.5 of pH, 10 mg of lipase AK dosage, 0.04 mmol
of substrate dosage and 40 h of reaction time. Under the optimum conditions, c and eep could reach up to 49% and 98%,
respectively.
Keywords Kinetic resolution · Lipase AK · Hydrolysis · 2-(4-Hydroxyphenyl) propionic acid ethyl ester
Introduction
selectivity, friendly to the environment and continuous pro-
duction by coupling technology (Saric et al. 2011), and it has
been widely applied in pharmaceutical industry.
Chiral drugs have taken up about one-third of the global
drug market. The market of optically pure drugs is increas-
ing dramatically, so the development of chiral separation
technology is crucial. Generally, the methods to obtain sin-
gle-enantiomer drugs mainly involve asymmetric synthesis
and racemic resolution. Asymmetric synthesis includes
fermentation, chiral source synthesis and asymmetric cata-
lytic synthesis (Schuur et al. 2011). Although asymmetric
synthesis has made impressive progress, racemic resolution
is still the most common way in industry production (Gha-
nem and Aboul-Enein 2004). A series of chiral resolution
methods have been developed, such as membrane separa-
tion (Weng et al. 2015), crystallization (Vetter et al. 2015),
chromatography (Shen and Okamoto 2015), and enantiose-
lective liquid–liquid extraction (ELLE) (Tang et al. 2011).
However, these methods have some shortages, such as low
capacity and high cost of chromatography (Lv et al. 2015),
low yield and versatility of crystallization (Wacharinean-
tar et al. 2010). Compared with above-mentioned meth-
ods, enzymatic resolution possesses the advantages of high
Lipase (Ec 3.1.1.3) belongs to carboxyl ester hydrolase,
which is employed as biocatalyst for various reactions such
as acylation (Li et al. 2012), esterifcation (Siodmiak et al.
2015), transesterifcation (Miyazawa and Iguchi 2013) and
hydrolysis (Leśniarek et al. 2018), etc. For example, lipase
from Candida rugosa, lipase from Pseudomonas cepacia
and lipase from Aspergillus terreus, can catalyze hydrolysis
of racemic arylpropionic acid drugs (Yilmaz et al. 2011;
Zhang et al. 2019; Hu et al. 2015). The preparation of opti-
cally pure compounds by lipase-catalyzed has recently
become a major research feld due to excellent catalytic
activity and high stereoselectivity of lipase.
2-Arylpropionic acid is an important class of anti-
infammatory drugs (NSAIDs) with antipyretic and analge-
sic efects. 2-(4-Hydroxyphenyl)propionic acid (2-HPPA)
is an intermediate of 2-arylpropionic acid drugs. Usually,
two enantiomers of NSAIDs difer greatly in their pharma-
cological activities (Gilani et al. 2017), thus the separation
of these drugs has important research signifcance. There
are many publications that have reported the separation and
synthesis of 2-arylpropionic acid (Neumann et al. 2010;
Ye et al. 2010). Tong et al. have reported the separation
of ( )-2-HPPA by HPLC (Tong et al. 2016), while enzy-
matic resolution has not been applied. In this paper, opti-
cally pure (R)-(−)-2-HPPA was obtained by lipase-catalyzed
* Kewen Tang
tangkewen@sina.com
1
Department of Chemistry and Chemical Engineering, Hunan
Institute of Science and Technology, Yueyang 414006,
Hunan, China
Vol.:(0123456789)
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Chemical Papers
hydrolysis of 2-HPPAEE enantiomers. Efects of diferent
reaction conditions on lipase activity and enantioselectivity
were investigated. Under the optimal conditions, 2-HPPAEE
enantiomers could be separated and high purity of (R)-(−)-
2-HPPA was obtained.
The enantioselectivity (E) is determined by the conver-
sion of 2-HPPAEE (c, %) and the enantiomeric excess of the
product (eep, %).
[(−)-acid] − [(+)-acid]
eep =
× 100%,
(1)
[(−) − acid] + [(+)-acid]
([(−)-acid] + [(+)-acid])V
c =
× 100%,
(2)
n2−HPPAEE,0
Experimental
Enzymes
ln[1 − c(1 + eep)]
E =
,
(3)
ln[1 − c(1 − eep)]
Lipozyme from Rhizomucor miehei (Lipozyme RM,
250 IUN/g), lipase from Rhizopus niveus (RNL, 2500 U/g)
and lipase from porcine pancreas (PPL, 20,000 U/mg) were
purchased from Sigma-Aldrich company (USA). Novo-
zym 40086 is lipase from Aspergillus oryzae (275 IUN/g),
Lipozyme TL IM is lipase from Thermomyces lanuginosus
(250 IUN/g) and CALA is lipase A from Candida antarc-
tica (6000 LU/g), which were purchased from Novozymes
Biopharma DK A/S (Denmark). Lipase AK is lipase from
Pseudomonas fuorescens (20,000 U/g) and Lipase AYS
(30,000 U/g) is lipase from Candida rugosa, which were
obtained from Amano Pharmaceutical company (Nagoya,
Japan).
where [(−)-acid] and [(+)-acid] represent the concentration
of (−)-2-HPPA and (+)-2-HPPA, respectively; V is the vol-
ume of reaction mixture; n2-HPPAEE,0 is the initial amount of
2-HPPAEE (mmol).
Synthesis of 2‑HPPAEE
2-HPPAEE was synthesized by esterifcation of commer-
cially available ( )-2-HPPA and ethanol. ( )-2-HPPA
(3.32 g, 20 mmol) and p-toluenesulfonic acid (0.86 g,
5 mmol) were dissolved in ethanol (29 mL, 800 mmol). The
mixture was added to a round-bottomed fask equipped with
a condenser tube and stirred for 12 h at 80 °C in an elec-
trothermally heated oil bath. Reaction was monitored with
TLC plate until the esterifcation reaction was terminated.
To remove the remaining substrate and p-toluenesulfonic
acid, the saturated sodium bicarbonate solution was applied
to wash-reactive mixture. The mixture was extracted with
ethyl acetate and then washed with deionized water to neu-
tral. Anhydrous sodium sulfate was used to dry the separated
organic phase and then concentrated by distillation under
reduced pressure (40 °C, 20 kPa). The spectroscopic data
of product are in accordance with literature (Ushiyama and
Furuya 1989). Finally, the racemic 2-HPPAEE (yield,≥90%;
purity,≥98%) was obtained as slight yellow oil.
Chemicals
Sulfobutylether-β-cyclodextrin (SBE-β-CD) was obtained
from Shandong Zhiyuan Biotechnology Co., Ltd (Shandong,
China). ( )-2-HPPA was obtained from Xianju Pharmaceu-
tical Co., Ltd (Zhejiang, China). 2-HPPAEE was prepared
in the laboratory. Solvent for chromatography was of HPLC
grade. All other reagents were analytical grade and obtained
from diferent commercial suppliers.
HPLC analysis
The concentrations of resolution products, (S)-(+)-2-HPPA
and (R)-(−)-2-HPPA were analyzed on reversed-phase col-
umn of ODS-3 flled with silica gel (250 mm×4.6 mm i.d.,
5 μm) by HPLC (waters e2695, Waters Corporation, USA).
UV detection wavelength was set at 230 nm. The mobile
phase was composed of methanol and aqueous solution
(containing 25 mmol/L SBE-β-CD and 0.5% glacial acetic
acid) volume ratio of 15:85 (pH 3.50, adjusted with trieth-
ylamine). The fow rate of the mobile phase was kept at
1.0 mL/min. The injection volume was 10 µL. The retention
time of (S)-(+)-2-HPPA, (R)-(−)-2-HPPA, (S)-(+)-2-HP-
PAEE and (R)-(−)-2-HPPAEE were 10.87, 12.96, 95.38, and
99.91 min, respectively. This analytical method was adjusted
by the literature (Tong et al. 2016).
Enzymatic hydrolysis of ( )‑2‑HPPAEE
The enzymatic hydrolysis reaction operated in a heated mag-
netic stirred reactor (IKA RCT basic, Germany) equipped
with 25 mL schlenk tube. The agitation speed was kept
400 rpm. A certain amount of ( )-2-HPPAEE and lipase
were added in 2 mL of 0.1 mol/L dibasic sodium phosphate
bufer to initialize the reaction. The hydrolysis reaction is
illustrated in Scheme 1. After the reaction was completed,
1 mL of acetonitrile was added to the reaction solution to
dilute and dissolve all the substrates and products. Then,
0.45 µm microporous membrane was used to flter the reac-
tion mixture. The concentrations of 2-HPPA enantiomers
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Chemical Papers
Scheme 1 Lipase-catalyzed enantioselective hydrolysis of ( )-2-HPPAEE in bufer medium
Table 1 Results of hydrolysis of ( )-2-HPPAEE catalyzed by difer-
enantioselectivity, while the catalytic activity of them is
ent lipases
not high. Lipase AK showed the highest E value (164) and
eep (> 98%). This may be because the substrate specifcity
of lipase determines that lipase AK prefers to form a com-
plex with (R)-(−)-2-HPPAEE compared to other lipases.
Therefore, lipase AK was chosen as the best biocatalyst
for all further experiments based on the results of activity
and selectivity.
Enzyme species Source
eep (%) c (%) E
Lipase AYS
CALA
Lipase AK
Lipozyme RM
Candida rugosa
Candida antarctica
Pseudomonas fuorescens 98
85
29
4
21
34
4
13
2
164
2
Rhizomucor miehei
9
Novozym 40086 Aspergillus oryzae
40
4
1
Lipozyme TL
PPL
RNL
Thermomyces lanuginosus 26
2
2
Porcine pancreatic
–
–
–
–
–
–
Efect of temperature
Rhizopus niveus
As we all know, in biocatalytic stereochemical reaction, tem-
perature has a great impact on enzyme activity, enantiose-
lectivity and reaction rate. Too low temperature will inhibit
enzyme activity, while too high temperature will make the
enzyme lose activity. Therefore, it is necessary to fnd the
optimum temperature of lipase AK-catalyzed hydrolysis of
( )-2-HPPAEE reaction. As shown in Fig. 1a, b, c, ees, eep
and E increased rapidly with the increase of temperature
(≤45 °C), and then they decreased with a further increase
of temperature (≥45 °C). Therefore, the maximum c (26%),
ees (35%), eep (98%) and E (161) are obtained at 45 °C. The
decrease of c, ees, eep and E in high temperature might be
explained by the reason that high temperature could dam-
age the three-dimensional structure of lipase AK and lead
to the loss of the activity. Furthermore, the excessive high
temperature could result in the conformational change of
the active site (Dong et al. 2010). When the temperature
decreases (≤45 °C), the energy barrier of two enantiomers
decreases, resulting in the reduction of enantioselectivity.
On the other hand, the collision chance between enzyme and
substrate molecules decreased which might be detrimental to
form enzyme–substrate complexes and then the reaction rate
was reduced at lower temperature. To maintain the activity
and selectivity of the lipase, an appropriate temperature was
needed. Therefore, the reaction temperature was set at 45 °C.
Conditions: reaction temperature 45 °C, pH 6.50, 10 mg lipase,
0.04 mmol ( )-2-HPPAEE, 24 h, and reaction volume 2 mL
were analyzed by HPLC. All the experiments of this paper
were repeated three times under identical conditions and
the precision level of the replicated values was within 3%.
Results and discussion
Selection of various lipases
The catalytic efficiency of different lipases may dif-
fer greatly in enantioselective hydrolysis of racemic
2-HPPAEE. Therefore, screening of lipases is necessary
to prioritize studies of other reaction conditions. The ste-
reoselectivity and activity of nine lipases were evaluated
by the conversion and enantiomeric excess under identical
conditions (Table 1). Table 1 shows the results of con-
version of substrate (c), enantiomeric excess of product
(eep) and enantioselectivity (E) of each lipase after 24 h of
reaction. Lipozyme RM, PPL and RNL are essentially una-
ble to catalyze the hydrolysis of ( )-2-HPPAEE. Lipase
AYS, Novozym 40086 and Lipozyme TL have a certain
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Chemical Papers
Fig. 1 Efect of temperature on c, ees, eep and E. Conditions: pH 5.5, 10 mg lipase AK, 0.04 mmol ( )-2-HPPAEE, reaction time 6 h, and reac-
tion volume 2 mL
acid residue (Arpigny and Jaeger 1999). In catalytic process,
the catalytic triad is converted into a tetrahedral transition
state by hydrogen bonding with the substrate, which is a key
step in determining the reaction rate and selectivity. Since
lipase is a catalytically active biomacromolecule, the tertiary
structure of the enzyme is destroyed at too high or too low
pH, which leads to the loss of catalytic activity (Cho et al.
2011; Dong et al. 2010). Meanwhile, due to the destruc-
tion of the three-dimensional structure of the enzyme, the
active site was reduced and then the initial reaction rate and
enantioselectivity were decreased. Similar results have been
reported in enantioselective hydrolysis of (R/S)-naproxen
methyl ester using lipase catalyst (Erdemir and Yilmaz
2012). Based on the above results, pH of 5.5 was selected
as the optimum pH.
Efect of pH
The pH of bufer solution has a great efect on lipase-cata-
lyzed hydrolysis reaction. Generally, lipases have an optimal
pH, and higher and lower values can lead to partial inactiva-
tion. Therefore, it is very important to fnd the optimum pH
for lipase-catalyzed hydrolysis of ( )-2-HPPAEE reaction.
Figure 2a, b depicts the infuence of pH on c, ees, eep and
E. It is observed from Fig. 2 that c, eep and E increased
with increasing the pH of bufer solution ranging from 4.0
to 5.5. With a further increase of pH, ees, c and E decreased
rapidly, while eep decreased slightly. The maximum values
of c (22%), ees (28%), and E (161) were obtained at pH 5.5.
Lipases have been reported to possess similar “catalytic tri-
plet” residues constructed from His, Ser and another amino
Fig. 2 Efect of pH on c, ees, eep and E. Conditions: reaction temperature 45 °C, 10 mg lipase AK, 0.04 mmol ( )-2-HPPAEE, reaction time 6 h,
and reaction volume 2 mL
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Chemical Papers
( )-2-HPPAEE (substrate) dosage on c, ees and eep were
investigated. As shown in Fig. 4, ee of product (eep) is very
high (approximately 98%) and can keep unchanged in the
tested dosage of substrate. The conversion is very high
(approximately 50%) with a relatively low substrate dos-
age (≤0.04 mmol) and then decreases with increasing the
substrate dosage (≥0.04 mmol). The increase of substrate
dosage also causes the decrease of ees. The above phe-
nomenon may be explained as follows: when the substrate
dosage is low (≤ 0.04 mmol), enzyme only binds to (R)-
(−)-HPPAEE to form an enzyme–substrate complex, which
leads to high ee of product. When the substrate dosage is
increased (≥0.04 mmol), an inhibition occurs, which leads
to the reduction of conversion rate and ees (Takaç and Mutlu
2007). Meanwhile, to obtain excellent conversion and ees,
Efect of lipase AK dosage
In lipase-catalyzed stereochemical reaction, increasing
the dosage of enzyme used can shorten the reaction time
required for the same conversion, but at the same time it
will increase the production cost of the process. Hence, it
is needed to determine an appropriate enzyme dosage. Fig-
ure 3a, b shows the efect of lipase AK dosage on c, ees, eep
and E. As shown in Fig. 3a, c and ees increase rapidly with
increasing the lipase AK dosage in the bufer containing
0.04 mmol of the dosage of substrate. When the enzyme
dosage reaches 10 mg, they remain unchanged with a further
increase of the lipase AK dosage. In addition, eep has no
signifcant efect as the amount of enzyme increases. From
Fig. 3b, E increases with the increase of enzyme dosage
(≤10 mg), and further increase of enzyme dosage will lead
to a signifcant downward trend (≥10 mg). The results show
that E is decreased from 330 to 266 with the increase of
enzyme dosage (≥10 mg). This may be attributed to the use
of free enzymes as catalysts to hydrolyze HPPAEE in phos-
phate bufers. The enzyme is appears easily in an intense
aggregation tendency in such a reaction system (Palomo
et al. 2003; Marciello et al. 2012). The E decreased with the
increase of enzyme dosage, which may be due to the mutual
aggregation of enzyme molecules at a higher concentration.
This result is consistent with that has been reported in the
literature (Takaç and Mutlu 2007). Therefore, lipase AK
dosage of 10 mg was selected with the substrate dosage of
0.04 mmol.
Efect of substrate dosage
Fig. 4 Efect of substrate dosage on c, ees and eep. Conditions: reac-
tion temperature 45 °C, pH 5.5, 10 mg lipase AK, reaction time 40 h
and reaction volume 2 mL
The dosage of substrate in the enzymatic reaction is a
key factor affecting its potential application. Effects of
Fig. 3 Efect of the dosage of lipase AK on c, ees, eep and E. Conditions: reaction temperature 45 °C, pH 5.5, 0.04 mmol ( )-2-HPPAEE, reac-
tion time 40 h and reaction volume 2 mL
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slow-reacting enantiomers. To meet the eep value of 98% and
c of 49%, the reaction time required was approximately 40 h.
Application
The optimal conditions were determined by the above
experiment’s results, involving temperature of 45 °C, pH
of 5.5, 10 mg lipase AK dosage, 0.04 mmol substrate dos-
age and 40 h of reaction time. Enantioselective hydrolysis
of HPPAEE enantiomers were carried out by lipase cataly-
sis under these optimal conditions. The purity of racemic
2-HPPA and obtained products were analyzed through
HPLC. Figure 6a shows the chromatograms of racemic
2-HPPA. Figure 6b shows the chromatograms of the prod-
ucts of hydrolysis of ( )-2-HPPAEE, indicating that high
purity of 98% is obtained.
Fig. 5 Efect of reaction time on c, ees and eep. Conditions: reac-
tion temperature 45 °C, pH 5.5, 10 mg lipase AK, 0.04 mmol
( )-2-HPPAEE and reaction volume 2 mL
the enzyme loading also needs to be increased. But com-
mercial lipases are relatively expensive, the economic ben-
efts are reduced. Therefore, considering the above reasons,
substrate dosage of 0.04 mmol in 2 mL of reaction system
was selected.
Conclusions
Stereoselective lipase-catalyzed hydrolysis of 2-HPPAEE
enantiomers was investigated to obtain (R)-(−)-2-HPPA.
Lipase AK was selected as the most efcient catalyst. The
efects of some important process parameters, including
temperature, pH, dosage of lipase AK, dosage of substrate
and reaction time were studied. It could be concluded that
the enantiomeric excess of product mainly depends on the
reaction temperature. The conversion of substrate primarily
depends on temperature, pH, dosage of lipase AK, reaction
time and dosage of substrate. The optimal conditions were
achieved, including temperature of 45 °C, pH of 5.5, 10 mg
lipase AK dosage, 0.04 mmol substrate dosage and reaction
time of 40 h, where the conversion could reach up to 49%
and the enantiomeric excess of product could reach up to
98%. A simple and feasible method is provided to obtain
optically pure (R)-(−)-2-HPPA by this work.
Process curve of enantioselective hydrolysis
of ( )‑2‑HPPAEE
Reaction time is one of the important factors guiding indus-
trial applications. When enzyme is at maximum activ-
ity, the required optimum reaction time will be reduced.
Therefore, infuences of reaction time on c, ees and eep
were investigated. From Fig. 5, c and ees increased rapidly
with the reaction time increase (≤40 h) and then increased
slowly (≥40 h), while the change in eep is small. It might
be because the reaction is highly enantioselective and only
one enantiomer of HPPAEE is hydrolyzed, at around 50%
conversion the reaction gets slower because in the mixture
there is little amount of fast-reacting enantiomer and a lot of
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Fig. 6 HPLC chromatograms.
a Racemate of ( )-2-HPPA,
b products of hydrolysis of
( )-2-HPPAEE
Phenom Macro Chem 72:189–196. https://doi.org/10.1007/s1084
7-011-9962-1
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Grant numbers, 21676077).
Ghanem A, Aboul-Enein HY (2004) Lipase-mediated chiral resolu-
tion of racemates in organic solvents. Tetrahedron Asymmetry
15:3331–3351. https://doi.org/10.1016/j.tetasy.2004.09.019
Gilani SL, Najafpour GD, Heydarzadeh HD, Moghadamnia A (2017)
Enantioselective synthesis of (S)-naproxen using immobilized
lipase on chitosan beads. Chirality 29:304–314. https://doi.
org/10.1002/chir.22689
Compliance with ethical standards
Conflict of interest The author declares that they have no confict of
interest.
Hu C, Wang N, Zhang W et al (2015) Immobilization of Aspergillus
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