Efficient resolution of 3-aryloxy-1,2-propanediols using CLEA-YCJ01 with high enantioselectivity

Article abstract of DOI:10.1039/c9ra01103j

The lipase YCJ01 from Burkholderia ambifaria is an organic solvent-stable enzyme and its activity can be activated by a hydrophobic solvent due to the "interface activation" mechanism. The activity of lipase YCJ01 increased by 2.1-fold with t-butanol as the precipitant even after cross-linking. The cross-linked enzyme aggregates of lipase YCJ01 (CLEAs-YCJ01) were found to be efficient for resolving 3-(4-methylphenoxy)-1,2-propanediol (MPPD) through sequential esterification. Excellent enantioselectivity towards MPPD (E > 400), excellent enantiomeric excess (ee) values of 99.2% for S-diacetates and 99.1% for R-monoacetate, and high yield (49.9%) were achieved using a high substrate concentration (180 mmol L^-1). Thus, R- and S-type compounds with excellent ee values were simultaneously obtained, and MPPD was resolved by CLEAs-YCJ01. CLEAs-YCJ01 also showed high operational stability and maintained 91.2% residual activity after ten batches. To further evaluate the substrate specificity of CLEAs-YCJ01, a series of 3-aryloxy-1,2-propanediols (six analogues of MPPD) was applied as substrates for resolution. Under the optimized reaction conditions of reaction temperature of 35 °C, MPPD concentration of 180 mmol L^-1, molar ratio of vinyl acetate to MPPD of 3 : 1, and isopropyl ether as the solvent, CLEAs-YCJ01 exhibited relatively strict enantioselectivity towards all the analogues of MPPD with a high yield (≥49.3%), favourable ee values (94.8-99.4%) for S-diacetates, and high ee values (92.1-99.2%) for R-monoacetate, which shows potential prospects for industrial applications.

Full text of DOI:10.1039/c9ra01103j

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Efficient resolution of 3-aryloxy-1,2-propanediols
using CLEA-YCJ01 with high enantioselectivity†
Cite this: RSC Adv., 2019, 9, 13757
ab
a
a
*
Bin Wu and Bingfang He
Bin Wang,
The lipase YCJ01 from Burkholderia ambifaria is an organic solvent-stable enzyme and its activity can be
activated by a hydrophobic solvent due to the “interface activation” mechanism. The activity of lipase
YCJ01 increased by 2.1-fold with t-butanol as the precipitant even after cross-linking. The cross-linked
enzyme aggregates of lipase YCJ01 (CLEAs-YCJ01) were found to be efficient for resolving 3-(4-
methylphenoxy)-1,2-propanediol (MPPD) through sequential esterification. Excellent enantioselectivity
towards MPPD (E > 400), excellent enantiomeric excess (ee) values of 99.2% for S-diacetates and 99.1%
for R-monoacetate, and high yield (49.9%) were achieved using a high substrate concentration
(180 mmol Lꢀ1). Thus, R- and S-type compounds with excellent ee values were simultaneously obtained,
and MPPD was resolved by CLEAs-YCJ01. CLEAs-YCJ01 also showed high operational stability and
maintained 91.2% residual activity after ten batches. To further evaluate the substrate specificity of
CLEAs-YCJ01, a series of 3-aryloxy-1,2-propanediols (six analogues of MPPD) was applied as substrates
for resolution. Under the optimized reaction conditions of reaction temperature of 35 ꢁC, MPPD
concentration of 180 mmol Lꢀ1, molar ratio of vinyl acetate to MPPD of 3 : 1, and isopropyl ether as the
solvent, CLEAs-YCJ01 exhibited relatively strict enantioselectivity towards all the analogues of MPPD
with a high yield ($49.3%), favourable ee values (94.8–99.4%) for S-diacetates, and high ee values (92.1–
99.2%) for R-monoacetate, which shows potential prospects for industrial applications.
Received 12th February 2019
Accepted 14th March 2019
DOI: 10.1039/c9ra01103j
rsc.li/rsc-advances
preparation of chiral APPDs, namely enzymatic methods, is
becoming increasingly attractive. Several epoxides at a 10 mM
Introduction
concentration were resolved to produce S-3-benzoxyl-1,2-
propanediols using an epoxide hydrolase derived from Asper-
gillus niger,8 and the enantioselectivity values ranged between
22 and 60 aer 2 h. In another study, a recombinant epoxide
hydrolase from Yarrowia lipolytica9 was applied as a catalyst for
the preparation of S-3-phenoxy-1,2-propanediols, and the ee
values of the products were 95%. However, water-dependent
side reactions such as the non-catalytic hydrolysis of epoxides
resulted in a decrease in the ee value and yield of the
product.10,11 On the other hand, most epoxide hydrolases pref-
erentially hydrolyze racemic epoxides into the S conguration of
the formed diol, which limits their applications in the prepa-
ration of R-type diols.
Lipases (esterases) are oen applied for the synthesis,
hydrolysis, and transesterication of esters and exhibit enan-
tioselective properties.12 For example, the dicarboxyesters of 1,2-
propanediols were sequentially hydrolyzed by an esterase13 to
obtain (S)-1-phenyl-1,2-ethanediol with an ee of 96%. Also,
chiral 3-aryloxy-1,2-propanediols can be directly obtained
through sequential esterication with a lipase. Theil's group14,15
used Amano lipase PS to separate 3-aryloxy-1,2-propanediols,
yielding R-type and S-type compounds with ee values in the
range of 55–99.8% and 79–96.8%, respectively, resulting in
moderate enantioselectivity towards the S-type compounds. The
Enantiopure 3-aryloxy-1,2-propanediols (APPDs) comprise
a class of important precursors for chiral drugs and pesticides.
S-type APPDs are synthetic intermediates for the synthesis of
cardiac propranolol, which is a drug for the treatment of
cardiovascular diseases.1,2 They can also be used to synthesize
phosphodiesterase inhibitors3,4 for the treatment of heart
disease. Furthermore, R-type APPDs are precursors for
a synthetic insect growth regulator.5,6 Therefore, considerable
research has been performed on preparation methods for chiral
APPDs, which can be divided into two categories.
The rst category comprises chemical methods. The enan-
tioselective esterication of the secondary hydroxyl group needs
a chiral chemical catalyst. Chiral nanoporous metal–metal-
losalen frameworks7 are used as catalysts to produce R-3-
benzoxy-1,2-propanediols, and the ee values of the products
range from 87% to 99%. However, these frameworks have
complex structures and their preparation includes complex
steps. Therefore, the second category of methods for the
aCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,
Nanjing 211816, China. E-mail: bingfanghe@njut.edu.cn
bSchool of Chemistry & Environmental Engineering, Jiangsu University of Technology,
Changzhou 213001, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra01103j
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lipase PS is hard to apply in the preparation of S-type YCJ01 are shown in Table 1. The type of precipitant used has
compounds. a great inuence on the activity recovery of CLEAs-YCJ01. When
The immobilization of the enzyme affords improved opera- saturated ammonium sulfate solution was used as the precipi-
tional stability and allows its facile separation and reuse.16 tant, the activity recovery of CLEAs-YCJ01 was 77.9%; however,
Thus, effective methodologies for enzyme immobilization as when the precipitant was changed to organic solvents, the
CLEAs (cross-linked enzyme aggregates) are applicable to activity recovery of CLEAs-YCJ01 was higher than 100%.
a broad range of enzymes, and CLEAs are proven to be signi- Specially, 214.8% of activity recovery was achieved using t-
cantly more stable to denaturation by heat, organic solvents and butanol as the precipitant. These results may be closely related
proteolysis than the corresponding soluble enzyme or lyophi- to the “interface activation” mechanism of lipase. Most lipases
lized (freeze-dried) powder.17 In this work, CLEAs of lipase have an activity center featuring an a-helical amphipathic
YCJ01 from Burkholderia ambifaria18 (CLEAs-YCJ01) were “lid”.19 In a hydrophobic environment or on an oil–water
prepared and evaluated for the resolution of 3-(4- interface, the lid is opened outwards from the active center so
methylphenoxy)-1,2-propanediol (MPPD). CLEAs-YCJ01 showed that it does not hinder the entry of the substrate into the active
strict enantioselectivity towards MPPD and excellent ee values center and the lipase shows an active form. In this study, the
were simultaneously obtained for both R- and S-type organic solvents used as precipitants could trigger the “inter-
compounds. A series of 3-aryloxy-1,2-propanediols was also face activation” mechanism. In the solvent t-butanol with
used to evaluate the substrate specicity of CLEAs-YCJ01.
higher hydrophobicity, the activity of lipase YCJ01 may be
activated efficiently and the active form was kept aer cross-
linking. Moreover, the solvent t-butanol with higher hydro-
phobicity may induce the aggregation of enzyme proteins in
a shorter time, which reduced the loss of enzymatic activity.20
Glutaraldehyde is the most commonly used cross-linker for
the preparation of CLEAs. The effect of glutaraldehyde
concentration on the activity recovery of CLEAs-YCJ01 is shown
in Table 2. As the glutaraldehyde concentration increased from
0.05% (v/v) to 0.25% (v/v), the activity recovery reached a high
level (214.8%). Further increasing the amount of glutaraldehyde
caused a great loss in the activity of CLEAs-YCJ01 and a low
activity recovery. Therefore, 0.25% was selected as the optimum
glutaraldehyde amount.
Results and discussion
Selection of precipitating agent for preparation of CLEAs-YCJ01
The selection of an appropriate precipitating agent is very
important for the preparation of CLEAs-YCJ01. According to the
previous study on the stability of lipase YCJ01 in various organic
solvents (25%, v/v) by Yao,18 the lipase YCJ01 is stable in
ethanol, 1-propyl alcohol, 2-propyl alcohol, t-butanol and
acetone. In addition, the commonly used protein precipitant,
saturated ammonium sulfate solution, was considered. There-
fore, these solvents were selected as the precipitants in our
present study. Their effects on the activity recovery of CLEAs-
Table 1 Special activity and activity recovery of CLEAs-YCJ01 in different precipitantsc
Precipitant
Specic activitya (ꢂ104 U gꢀ1
)
Activity recoveryb (%)
Ethanol
1-Propanol
2-Propanol
Acetone
t-Butanol
Saturated ammonium sulfate
solution
4.44 ꢃ 0.20
5.26 ꢃ 0.30
5.63 ꢃ 0.24
4.83 ꢃ 0.32
10.86 ꢃ 0.37
3.44 ꢃ 0.17
100.6 ꢃ 4.6
119.1 ꢃ 6.9
127.5 ꢃ 5.5
109.3 ꢃ 7.2
214.8 ꢃ 8.3
77.9 ꢃ 3.8
a
Special activity ¼ (activity in the CLEAs/weight of CLEAs) ꢂ 100%. b Activity recovery ¼ (activity in the CLEAs/activity of lipase solution used) ꢂ
100%. c Conditions: protein concentration of enzyme solution: 4.0 mg mLꢀ1, activity of lipase YCJ01: 176.6 U mLꢀ1, volume of precipitant : volume
of enzyme solution ¼ 3 : 1, cross-linking time ¼ 4 h and glutaraldehyde amount ¼ 0.25% (v/v, %).
Table 2 Effect of the amount of glutaraldehyde on CLEAs-YCJ01 activity
Amounta (v/v, %)
Specic activitya (ꢂ104 U gꢀ1
)
Activity recoveryb (%)
0.05
0.15
0.2
0.25
0.3
13.02 ꢃ 0.46
11.87 ꢃ 0.28
11.53 ꢃ 0.37
10.86 ꢃ 0.37
9.99 ꢃ 0.37
7.97 ꢃ 0.48
6.12 ꢃ 0.41
165.8 ꢃ 10.4
192.3 ꢃ 6.3
211.3 ꢃ 8.4
214.8 ꢃ 8.3
202.8 ꢃ 8.4
166.4 ꢃ 10.8
130.0 ꢃ 9.3
0.35
0.375
a
b
Amount of glutaraldehyde ¼ glutaraldehyde volume/enzyme volume ꢂ 100%. Reaction conditions: enzyme protein concentration ¼ 4.0 mg
mLꢀ1, activity of enzyme solution ¼ 176.6 U mLꢀ1, volume of t-butanol : volume of enzyme ¼ 3 : 1, and cross-linking time ¼ 4 h.
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microenvironment of the active center, which hindered the
proper orientation of the substrates in the active center and led
to a reduction in the reaction rate.23 Thus, a solvent with
moderate hydrophobicity is benecial to maintain the “critical
water” of the enzymatic surface and also facilitate the proper
orientation of substrates in its active center.
The values of ee for the S-diacetates were determined and also
presented in Table 3. The solvents markedly affected the enan-
tioselectivity of CLEAs-YCJ01. Fortunately, in isopropyl ether,
CLEAs-YCJ01 not only showed the highest activity, but also the
highest enantioselectivity towards MPPD. When the hydrophobic
solvents hexane and toluene were used as solvents, the enantio-
selectivity was 126 and 89, respectively. When diisopropyl ether
was used as the solvent, the enantioselectivity rapidly increased
to 1190. However, upon changing the solvents to the hydrophilic
solvents t-butanol, THF and acetone, the enantioselectivity
decreased to 105, 24 and 37, respectively. Clearly, isopropyl ether
proved to be the most suitable solvent for the resolution of MPPD
through transesterication. Isopropyl ether also favored the
Penicillium roqueforti lipase24 and porcine pancreatic lipase,25
which exhibited the best catalytic performances for the resolu-
tion of secondary alcohols through transesterication. However,
the reason for these lipases showing their best catalytic perfor-
mances in isopropyl ether is unclear. The enantioselectivity of
lipases may change, sometimes dramatically, as a function of the
nature of the solvents and for other reasons.26
Effect of the molar ratio of vinyl acetate to MPPD on the
resolution of MPPD. The molar ratio of vinyl acetate to MPPD is
a key factor in the industrial application of enzymes. Thus, its
effects on the conversion and enantioselectivity were investi-
gated and the results are shown in Table 4. When the molar
ratio of vinyl acetate to MPPD was increased from 2 : 1 to 3 : 1,
the conversion of MPPD increased rapidly from 40.1% to 49.3%
in 9 h. This is because an increased abundance of acyl donors
will contribute to the formation of acyl-enzyme intermediates,
thus speeding up the reaction rate.27 With a further increase in
the molar ratio of vinyl acetate to MPPD, there was no further
increase in the conversion and the E-values still maintained
a high level (E > 400). This means that an abundance of acyl
donors does not alter the enantioselectivity of CLEAs-YCJ01 to
MPPD. Therefore, the appropriate molar ratio of vinyl acetate to
MPPD is 3 : 1.
Scheme
propanediols.
1 Enzymatic resolution of racemic 3-aryloxy-1,2-
Optimization for the resolution of MPPD by CLEAs-YCJ01
As shown in the Scheme 1, the reaction was carried out with
vinyl acetate in the presence of CLEAs-YCJ01. 3-(4-
Methylphenoxy)-1,2-propanediol (MPPD, substrate 2) was rstly
formed into primary monoacetate, and the formed monoacetate
was enantioselectively converted into the S-diacetates. In the
next experiment, the conditions of resolution were optimized to
improve the reaction rate and ee values.
The effect of organic solvents on the resolution of MPPD. In
non-aqueous enzymology, medium engineering is an effective
means to control and regulate the structure and function of
enzymes.21 Thus, the effects of six solvents with different
hydrophobicities (log P, n-hexane/water distribution coefficient)
on the activity and enantioselectivity of CLEAs-YCJ01 were
investigated. As shown in Table 3, in the moderately hydro-
phobic isopropyl ether, the conversion of MPPD reached 49.1%;
however, in the hydrophilic solvents tetrahydrofuran (THF) and
acetone, the conversions were 41.6% and 43.2%, respectively. In
the hydrophilic solvents, the “critical water” layer at the enzy-
matic surface is stripped off by the solvent,22 resulting in an
increase in the rigidity of the enzyme structure and a decrease
in its reaction activity. In isopropyl ether, the “critical water”
layer on the enzymatic surface was maintained, which favored
the exibility of the enzyme, and thus the enzyme retained its
high activity. Meanwhile, in hexane and toluene with higher
hydrophobicity, the activity of CLEAs-YCJ01 decreased, which
may be because these highly hydrophobic solvents altered the
Table 3 Resolution of racemic MPPD in non–aqueous solvents
Catalyst
Solvent
log P
Initial rates (mmol minꢀ1
)
ee of S-diacetates
c (%)
Ea
Lipase YCJ01
Diisopropyl ether
t-Butanol
THF
1.9
0.6
0.49
ꢀ0.23
1.9
67.2 ꢃ 1.9
51.1 ꢃ 2.3
32.3 ꢃ 2.2
41.6 ꢃ 2.2
81.8 ꢃ 0.2
74.2 ꢃ 1.3
67.8 ꢃ 1.9
71.5 ꢃ 2.0
99.1 ꢃ 0.1
94.6 ꢃ 0.4
73.7 ꢃ 0.6
82.7 ꢃ 0.5
99.7 ꢃ 0.1
97.1 ꢃ 0.4
85.9 ꢃ 0.5
89.4 ꢃ 0.6
40.4 ꢃ 1.2
33.4 ꢃ 1.5
25.9 ꢃ 1.8
30.1 ꢃ 1.6
49.1 ꢃ 0.1
44.6 ꢃ 0.8
41.6 ꢃ 1.4
43.2 ꢃ 1.2
406 ꢃ 23
58 ꢃ 3
9 ꢃ 2
Acetone
15 ꢃ 2
CLEAs-YCJ01
Diisopropyl ether
t-Butanol
THF
1190 ꢃ 35
105 ꢃ 5
24 ꢃ 3
0.6
0.49
ꢀ0.23
Acetone
37 ꢃ 3
a
E ¼ ln[(1 ꢀ c)(1 ꢀ ees)/(1 ꢀ c)(1 + ees)], reaction conditions: 50 mM MPPD and 100 mM vinyl acetate was catalyzed in 20 mL solvent containing
20 mg of CLEAs-YCJ01, the mixture was incubated at 30 ꢁC and rotated at 180 rpm, and the reaction time was 12 h.
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Table 4 Effect of molar ratio of vinyl acetate to MPPD on conversion
and enantioselectivity
ee
Molar ratio R-Monoacetate S-Diacetates c (%)
Ea
2 : 1
2.5 : 1
3 : 1
3.5 : 1
4 : 1
66.6 ꢃ 0.3
85.2 ꢃ 0.2
96.4 ꢃ 0.2
96.1 ꢃ 0.2
96.5 ꢃ 0.2
99.7 ꢃ 0.1
99.6 ꢃ 0.1
99.4 ꢃ 0.1
99.3 ꢃ 0.1
99.4 ꢃ 0.1
40.1 ꢃ 0.2
46.1 ꢃ 0.2
730 ꢃ 30
900 ꢃ 30
49.3 ꢃ 0.2 1672 ꢃ 40
49.2 ꢃ 0.1 1220 ꢃ 35
49.2 ꢃ 0.1 1114 ꢃ 35
a
E ¼ ln[(1 ꢀ c)(1 ꢀ ees)/(1 ꢀ c)(1 + ees)], reaction conditions: 50 mM
MPPD was catalyzed in 20 mL isopropyl ether containing 20 mg
CLEAs-YCJ01, the mixture was incubated at 30 ꢁC and rotated at
180 rpm. The reaction time was 9 h.
Fig. 2 Effect of water content on the resolution of MPPD. Reaction
conditions: the resolution of MPPD was performed in 20 mL isopropyl
ether containing 50 mM MPPD, 150 mM vinyl acetate and 20 mg
CLEAs-YCJ01. The mixture was incubated at 35 ꢁC, rotated at 180 rpm
and the reaction time was 3 h.
The effect of temperature on the resolution of MPPD. The
effect of temperature on the enzymatic reaction was investi-
gated and the results are shown in Fig. 1. The catalytic activity
increased from 42.6 to 68.2 mmol (min g)ꢀ1 in the range of 30–
The effect of substrate concentration on the resolution of
MPPD. The effect of substrate concentration on the resolution
of MPPD is shown in Fig. 3. The reaction rate increased with an
increase in the substrate concentration. However, when the
substrate concentration exceeded 180 mmol Lꢀ1, the rate
decreased sharply from 246.5 to 182.7 mmol (min g)ꢀ1. There
may be two reasons for the decline in the reaction rate. First,
when the substrate concentration reached more than 180 mmol
Lꢀ1, MPPD was not completely dissolved in the solvent. Second,
a high concentration of formed monoacetate may increase the
viscosity of the reaction system, leading to a decrease in the
reaction rate. Meanwhile, the change in concentration had little
effect on the enantioselectivity of the enzyme, and the ee value
ꢁ
ꢁ
50 C. However, when the temperature exceeded 35 C, the ee
values of S-diacetates decreased to below 99%. High tempera-
ture conditions change the structure of the catalytic center of
lipases,28 which explains the reduction in the enantioselectivity
of CLEAs-YCJ01 towards MPPD at high temperatures. Thus, the
ꢁ
optimum reaction temperature was determined to be 35 C.
The effect of the water content of the solvent on the reso-
lution of MPPD. As shown in Fig. 2, CLEAs-YCJ01 showed high
catalytic activity when the water content was between 0.56 and
0.75, and the conversion reached more than 49% at 3 h.
However, the conversion at 3 h was less than 42% at the low
water contents of 0.11, 0.24, and 0.33. Additionally, the ee values
of the S-diacetates decreased slightly to 98.2% at a water content
of 0.75. The above results indicated that a high water content
improved the exibility and catalytic activity of CLEAs-YCJ01
while decreasing its enantioselectivity,29 leading to a decrease
in the ee values of S-diacetates. Therefore, the appropriate water
content is 0.56.
of S-diacetates was 99.2% at 180 mmol Lꢀ1
.
MPPD enzymatic reaction process curve. According to the
results for the resolution of MPPD with CLEAs-YCJ01, the
optimum conditions for enzyme catalysis included the use of
isopropyl ether as the solvent, MPPD concentration of
180 mmol Lꢀ1, molar ratio of vinyl acetate to substrate of 3 : 1,
Fig. 3 Effect of substrate concentration on the resolution of MPPD.
Fig. 1 Effect of temperature on the resolution of MPPD. Reaction Reaction conditions: the resolution of MPPD was performed in 20 mL
conditions: the resolution of MPPD was performed in 20 mL isopropyl isopropyl ether containing 20 mg CLEAs-YCJ01 and the molar ratio of
ether containing 50 mM MPPD, 100 mM vinyl acetate and 20 mg MPPD to vinyl acetate was 3 : 1. The mixture was incubated at 35 ꢁC,
CLEAs-YCJ01. The mixture was rotated at 180 rpm and the reaction rotated at 180 rpm with a water content 0.56, and the reaction time
time was 6 h.
was 6 h.
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reaction temperature of 35 ꢁC and water content of 0.56. Under were achieved in the rst batch. Thereaer, the activity of lipase
these optimum conditions, the resolution process curve of YCJ01 decreased. The residual activity of CLEAs-YCJ01 aer ten
MPPD with CLEAs-YCJ01 was obtained, as shown in Fig. 4. At batches remained at 91.2% of the initial activity, and the ee
the beginning of the reaction, S-monoacetate was rapidly values of S-diacetates remained above 99.0% aer every batch.
transformed into S-diacetate. As the reaction progressed These results showed that CLEAs-YCJ01 has good operational
further, the substrate concentration gradually decreased, which stability and the reaction time does not change the enantiose-
led to a slow increase in conversion. At the reaction time of 12 h, lectivity of CLEAs-YCJ01, indicating that the two congurations
the yield of S-diacetates was 49.9% and the ee values for mon- of substrate with high ee values could be achieved by prolong-
oacetate and S-diacetates were 99.1% and 99.2%, respectively. ing the reaction time or adding catalyst at every batch reaction.
Thus, excellent ee values were simultaneously obtained for both
The methods for the preparation of enantiomerically pure
the R- and S-type compounds, and the resolution of MPPD by MPPD include asymmetric catalysis and enzyme catalysis. Zhu
CLEAs-YCJ01 gave 49.9% yield for S-diacetates and 49.3 yield for et al.7 applied a chiral nanoporous metal–organic catalyst for
R-monoacetate.
the preparation of S-MPPD with a substrate concentration of
Operational stability of CLEAs-YCJ01. The operational 80 mmol Lꢀ1 and the reaction reached 57% conversion and 87%
stabilities of CLEAs-YCJ01 and lipase YCJ01 for the resolution of ee aer 24 h. Bendigiri et al.9 reported a recombinant epoxide
MPPD are shown in Fig. 5. When the reaction was catalyzed by hydrolase from Yarrowia lipolytica for the preparation of S-
CLEAs-YCJ01, the highest conversion and enantioselectivity MPPD and the ee was 95%. Zhao et al.30 applied an epoxide
hydrolase for the preparation of R-MPPD and the ee was 73%.
Theil et al.15 reported a transformation in the presence of
Amano lipase PS from B. cepacia, in which the yield of S-diac-
etates reached 42% and the ee for S-diacetates was 93%. In the
above studies, the concentration of substrate was in the range of
10–80 mmol Lꢀ1. In this work, a high enantioselectivity of S-
diacetates and substrate concentration were obtained using
CLEAs-YCJ01 as a catalyst. The ee values for monoacetate and S-
diacetates were 99.1% and 99.2%, respectively, and the
substrate concentration reached 180 mmol Lꢀ1, which shows
potential prospects for industrial applications.
Resolution of 3-aryloxy-1,2-propanediols by CLEAs-YCJ01
Fig. 4 Time course of resolution of MPPD by CLEAs-YCJ01. Reaction
Since CLEAs-YCJ01 showed excellent enantioselectivity towards
conditions: the resolution of MPPD was performed in 20 mL isopropyl
MPPD, it was evaluated for the resolution of a series of 3-aryloxy-
ether containing 180 mM MPPD, 540 mM vinyl acetate and 20 mg
CLEAs lipase YCJ01. The mixture was incubated at 35 ꢁC and rotated at
180 rpm with a water content of 0.56.
1,2-propanediols (six analogues of MPPD). The reactions are
shown in Scheme 1 and the results are shown in Table 5. Every
S-monoacetate of 3-aryloxy-1,2-propanediols preferentially
transformed into S-diacetates with a high conversion ($49.3%),
favorable ee values (94.8–99.4%) for S-diacetates and high ee
values (92.1–99.2%) for R-monoacetate. This means that CLEAs-
YCJ01 has relatively strict enantioselectivity towards all the
tested 3-aryloxy-1,2-propanediols. Especially, CLEAs-YCJ01 had
strict enantioselectivity towards 3-(4-methylphenoxy)-1,2-
propanediol (substrate 2, Table 5), and the ee values for mon-
oacetate and S-diacetates were 99.1% and 99.2%, respectively.
Also, 99.2% ee of monoacetate and 99.4% ee of S-diacetates
were obtained for 3-phenoxy-1,2-propanediol (substrate 1, Table
5) by CLEAs-YCJ01.
The six types of 3-aryloxy-1,2-propanediols have the common
structure of 3-phenoxy-1,2-propanediols with different substit-
uents on their benzene ring. As shown in Table 5, substrate 4
has an ortho benzene methyl moiety and substrate 6 has a para
Fig. 5 Operational stabilities of the lipase YCJ01 and CLEAs-YCJ01.
Reaction conditions: the resolution of MPPD was performed in 20 mL
isopropyl ether containing 180 mM MPPD, 540 mM vinyl acetate and benzene methyl moiety. CLEAs-YCJ01 showed different enan-
20 mg CLEAs-YCJ01. The mixture was incubated at 35 ꢁC and rotated
tioselectivities to these two substrates. Overall, the substrates
having a para substituent on the benzene ring did not greatly
at 180 rpm with a water content 0.56. Residual activity ¼ (the
conversion of MPPD/the conversion of MPPD for the first cycle) ꢂ
affect the enantioselectivity, while that having an ortho
100%. Operational conditions: the first cycle ended with the conver-
substituent showed a large enantioselective effect. It may be
sion of 49.2% for MPPD at 12 h, and the other cycles ended with the
same time of the first cycle.
possible that the ortho substitution of the substrate near the
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Table 5 Resolution of racemic 3-aryloxy-1,2-propanediols in non-aqueous solventsa
ee
No.
1
Substrate
Catalyst
Monoacetate
Diacetates
c (%)
E
Lipase YCJ01
CLEAs-YCJ01
78.5 ꢃ 1.0
99.2 ꢃ 0.1
99.1 ꢃ 0.1
99.4 ꢃ 0.1
44.2 ꢃ 0.3
49.9 ꢃ 0.1
529 ꢃ 12
1836 ꢃ 132
Lipase YCJ01
CLEAs-YCJ01
83.4 ꢃ 1.0
99.1 ꢃ 0.1
99.1 ꢃ 0.1
99.2 ꢃ 0.1
45.7 ꢃ 0.3
49.9 ꢃ 0.1
601 ꢃ 18
1347 ꢃ 78
2
Lipase YCJ01
CLEAs-YCJ01
57.8 ꢃ 1.0
95.2 ꢃ 0.1
96.2 ꢃ 0.6
96.0 ꢃ 0.2
37.5 ꢃ 0.3
49.8 ꢃ 0.1
94 ꢃ 18
184 ꢃ 21
3
4
Lipase YCJ01
CLEAs-YCJ01
44.8 ꢃ 1.6
92.1 ꢃ 0.2
94.4 ꢃ 0.7
94.8 ꢃ 0.2
32.2 ꢃ 0.6
49.3 ꢃ 0.1
54 ꢃ 9
124 ꢃ 11
Lipase YCJ01
CLEAs-YCJ01
60.4 ꢃ 1.4
95.2 ꢃ 0.2
95.4 ꢃ 0.5
95.3 ꢃ 0.2
38.8 ꢃ 0.4
49.9 ꢃ 0.1
78 ꢃ 12
156 ꢃ 11
5
Lipase YCJ01
CLEAs-YCJ01
74.5 ꢃ 1.8
95.6 ꢃ 0.2
96.7 ꢃ 0.1
96.5 ꢃ 0.2
43.5 ꢃ 0.7
49.8 ꢃ 0.1
137 ꢃ 16
219 ꢃ 17
6
a
Reaction conditions: 180 mM 3-phenoxy-1,2-propanediols and 540 mM vinyl acetate were catalyzed in 20 mL isopropyl ether containing 20 mg of
CLEAs-YCJ01 (or 43 mg of lipase YCJ01 powder with the same activity of CLEAs-YCJ01), the mixture was incubated at 35 ꢁC and rotated at 180 rpm
with a water content 0.56. The reaction was monitored by HPLC.
catalytic active center slightly affects the substrate orientation
Enzyme activity assay
in the active center, thus reducing the enantioselectivity of the
The hydrolytic activity of the free lipase was assayed according
to reported literature procedures and pNPP was used as the
substrate.31 CLEAs-YCJ01 was suspended in phosphate buffer
(pH ¼ 7.5, 50 mM) and samples of this suspension were used to
calculate its activity according to the procedure reported for the
free enzyme.
enzyme
towards
ortho-substituted
compound–lipase
complexes.15
Theil's group14,15 used Amano lipase PS to resolve 3-aryloxy-
1,2-propanediols. The ee values of R-type compounds ranged
from 55% to 99.8%, while that of S-type compounds ranged
from 79% to 96.8%. In summary, CLEAs-YCJ01 showed good
enantioselectivity towards 3-aryloxy-1,2-propanediols with high
ee values for both R- and S-type compounds, indicating that
CLEAs-YCJ01 has relatively strict substrate selectivity towards
all the used analogues of MPPD.
Transesterication reaction
The enzymatic transesterication reactions were performed in
50 mL conical asks with stoppers. The appropriate amounts of
solvent, 3-aryloxy-1,2-propanediols, vinyl acetate, and enzyme
were introduced into the conical asks and incubated at
a constant temperature and shaking at 180 rpm. Aliquots of
samples (30 mL) were withdrawn from the reaction mixture at
intervals and the samples were analyzed via high-performance
liquid chromatography (HPLC) according the following
method.
Experimental
Materials
The lipase YCJ01 from B. ambifaria was characterized in our
previous report, and its amino acid sequence was assigned
GenBank accession no. JQ733583. B. ambifaria YCJ01 was
deposited in CCTCC (Wuhan, China) with the accession
number CCTCC M 2011058.18 3-Aryloxy-1,2-propanediols were
Analytical methods
purchased from Heowns Biochemical Technology LLC (Tianjin, The reaction mixture was sampled at intervals and the solvent
China). p-Nitrophenyl palmitate (pNPP) was purchased from in each sample was volatilized and the remains were redissolved
Sigma-Aldrich (St. Louis, MO, USA). Vinyl acetate, isopropyl in a mobile phase composed of n-hexane/isopropanol (85/15, v/
ether and all other chemicals were purchased from Sinopharm v) (200 mL) then ltered using a 0.45 mm membrane lter. Aer
Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were ltration, the treated samples were analyzed via HPLC using
used directly without any further treatment.
a Chiralcel® OD-H column (Daicel, 4.6 ꢂ 250 mm) and detected
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at the ultraviolet wavelength of 270 nm. The mobile phase ow and the reaction time does not change the enantioselectivity of
rate was 1 mL minꢀ1 and 20 mL of each sample was injected at CLEAs-YCJ01, indicating that the two congurations of
30 ꢁC. The retention times of all the substrates are presented in substrate with high ee values could be achieved by prolonging
the ESI.†
The ee values of R-monoacetate (ees) and S-diacetates (eep)
the reaction time or adding catalyst at every batch reaction.
Further, CLEAs-YCJ01 was evaluated for the resolution of
were calculated by HPLC. The yield of S-diacetates was deter- a series of 3-aryloxy-1,2-propanediols (six analogues of MPPD)
mined by HPLC. The conversion of 3-aryloxy-1,2-propanediols under the optimized reaction conditions for the resolution of
(c) was calculated as c (%) ¼ ees/(ees + eep) ꢂ 100 and the MPPD. The results showed that CLEAs-YCJ01 has relatively
enantioselectivity (E) was calculated as E ¼ ln[(1 ꢀ c)(1 ꢀ ees)/(1 strict enantioselectivity towards 3-aryloxy-1,2-propanediols with
ꢀ c)(1 + ees)].
a high yield ($49.3%), favorable ee values (94.8–99.4%) for S-
diacetates and high ee values (92.1–99.2%) for R-monoacetate.
Especially, towards 3-(4-methylphenoxy)-1,2-propanediol and 3-
phenoxy-1,2-propanediol, both of R- and S-type compounds
with >99% ee were obtained.
Water content
The substrates and solvents were separately pre-equilibrated at
different water contents (aw) prior to the resolution. All samples
were placed in a closed vessel above saturated salts, and equi-
librium was reached for 48 h at room temperature. The salts
used in this test included LiCl, aw ¼ 0.11; CH3COOK, aw ¼ 0.24;
MgCl2, aw ¼ 0.33; Mg(NO3)2, aw ¼ 0.56, and NaCl, aw ¼ 0.75.
Conflicts of interest
There are no conicts to declare.
Preparation of CLEAs of lipase YCJ01 (CLEAs-YCJ01)
Acknowledgements
The preparation of CLEAs-YCJ01 in this work was done
according to the procedures described by Schoevaart20 with
some modications. Crude lipase YCJ01 powder was prepared
in our previous work18 and dissolved in phosphate buffer (pH ¼
7.5 and 0.05 M) to a concentration of 1.2 mg protein per mL (53
U). Aggregates of lipase YCJ01 were prepared using precipitants
and glutaraldehyde was added to a total concentration of 0.25%
(v/v), then the resulting suspensions were stirred at 0 ꢁC for 4 h.
The CLEAs of lipase YCJ01 was separated by centrifugation and
obtained by volatilizing the residual solvent. All portions of the
ltrate were retained for the determination of protein concen-
tration and activity.
This work was supported by the National High Technology
Research and Development Key Program of China
(2012AA022205).
Notes and references
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In a typical reaction, 20 mL isopropyl ether, CLEAs-YCJ01 (20
mg), MPPD (50 mM), and vinyl acetate (100 mM) were loaded
into 50 mL conical asks with stoppers. The mixture was kept at
35 ꢁC and rotated at 180 rpm. The samples were withdrawn
from the mixture and analyzed by HPLC, as described above.
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the ltered CLEAs-YCJ01 was washed with fresh isopropyl ether
(3 ꢂ 5 mL) and loaded back into the conical asks for the next
cycle.
Conclusions
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