Remarkably improved stability and enhanced activity of a: Burkholderia cepacia lipase by coating with a triazolium alkyl-PEG sulfate ionic liquid

Article abstract of DOI:10.1039/c7gc02319g

Three types of triazolium cetyl-PEG10 sulfate ionic liquid were synthesized and their activation of Burkholderia cepacia lipase was investigated; both the reaction rate and enantioselectivity depended on the cationic part of the coating ILs and 1-butyl-3-methyl-1,2,3-triazolium cetyl-PEG10 sulfate (Tz1)-coated lipase PS, which is especially suitable for the transesterification of 1-(pyridin-2-yl)ethanol, 1-(pyridin-3-yl)ethanol, and 1-(pyridin-4-yl)ethanol, among 12 types of tested secondary alcohol. The most important result was obtained when these enzymes were stored in an IL ([N_221MEM][Tf₂N]) solvent: Tz1-PS showed an amazing stability and it exhibited an excellent activity after 2 years when the enzyme was stored in [N_221MEM][Tf₂N].

Full text of DOI:10.1039/c7gc02319g

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ARTICLE
Remarkable improved stability and enhanced activity of a
Burkholderia cepacia lipase by coating with a triazolium alkyl-PEG
sulfate ionic liquid
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a, b
a, b
T. Nishihara, A. Shiomi, S. Kadotani, T. Nokami, T. Itoh
*
DOI: 10.1039/x0xx00000x
Three types of triazolium cetylꢀPEG10 sulfate ionic liquids were synthesized and their property for activation of
Burkholderia cepacia lipase investigated; both the reaction rate and enantioselectivity depended on the cationic part of the
coating ILs and 1ꢀbutylꢀ3ꢀmethylꢀ1,2,3ꢀtriazolium cetylꢀPEG10 sufate (Tz1)ꢀcoated lipase PS, especially suitable for the
transesterification of 1ꢀ(pyridinꢀ2ꢀyl)ethanol, 1ꢀ(pyridinꢀ3ꢀyl)ethanol, and 1ꢀ(pyridinꢀ4ꢀyl)ethanol, among 12 types of tested
www.rsc.org/
secondary alcohols. The most important result was obtained when these enzymes were stored in an IL ([N221MEM][Tf
solvent: TZ1ꢀPS showed an amazing stability and it exhibited an excellent activity after 2 years when the enzyme was stored
N].
2
N])
in [N221MEM][Tf
2
that
1ꢀbutylꢀ2,3ꢀdimethylimidazolium
3
,6,9,12,15,18,21,24,27,30ꢀdecaoxacetyltriacontyl sulfate
Introduction
6
,7
(cetylꢀPEG10 sulfate)
(
IL1
)
worked as an excellent
The value of the enzymatic reaction in organic synthesis is
highly respected from an environmentallyꢀfriendly aspect and
has now reached an industriallyꢀproven level in the
activating agent of Burkholderia cepacia lipase, and the
resulting immobilized enzyme (IL1-PS) by lyophilization
displayed an excellent reactivity toward many secondary
1
6,7
pharmaceutical and food industries. Lipases (triglycerol
alcohols in conventional organic solvents. Furthermore, the
acylhydrolases EC 3.1.1.3) are a group of hydrolases that are
some of the most widely used enzymes as a synthetic tool
because they can catalyse number of reactions including
hydrolysis, transesterification, alcoholysis, acidolysis,
recyclable use of the IL1-PS was realized when the reaction
was conducted in an ionic liquid solvent. Activation of lipase
8
by immobilization of the enzyme with ionic surfactant
compounds that have alkyl PEG moiety was also reported by
1
9
esterification, and aminolysis. Ionic liquids (ILs) have very
Kim and coꢀworkers. Rahman and coꢀworkers reported that
good properties as reaction media in chemical reactions;
they are lessꢀvolatile, lessꢀflammable, and have a low
toxicity and unique solubility for organic and inorganic
amino acid ILs also worked as good activating agent of
1
0
Candida rugose lipase through lyophilisation.
The
immobilization of enzymes by lyophilization with surfactants
or ILs might be a promising method of activation of
2
materials. ILs have now been used as reaction media for
4
,11
biotransformation,
reactions. Lipases are generally tolerable to a wide number
of substrates, however, very slow reactions or poor
in
particular,
lipaseꢀcatalyzed
lipases.
3
,4
During the study, we were noticed that the significant drop in
the enantioselectivity was caused when the lipase was
immobilized by IL2, which has an acidic proton at the 2ꢀ
1
enantioselective reactions are sometimes obtained. To solve
7
a
this problem, Kim and Lee first reported ionic liquid mediated
activation of enzyme focused on modified enantioselectivity
in 2002, though no acceleration had been accomplished by
position of the imidazolium ring. On the other hand, the
chirality of the substituent on the imidazolium cation also
significantly influenced the activation property of the ILs; 1ꢀ
5
their method. We reported the key solution to realize
((
R)ꢀpyrroridinꢀ2ꢀyl)methylꢀ2,3ꢀdimethylimidazolium cetylꢀ
6
improved performance of lipases using ILs in 2004; we found
PEG10 sulfate more strongly activated the lipase compared to
1
2
that of the (S)ꢀisomer. We then discovered a cooperative
activation of IL1 with amino acids; coating the lipase PS
using IL1 with LꢀProline or LꢀTyrosine effectively activated
the transesterification of secondary alcohols while maintaining
1
3
14
a perfect enantioselectivity (E value >200). Furthermore,
the tributyl(2ꢀmethoxyethyl)phosphonium cetylꢀPEG10
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ARTICLE
Journal Name
1
5
sulfate (PL1)ꢀcoated lipase PS (PL1-PS
)
showed a different
enzyme protein; this was the same in the case of the IL1 when 1ꢀ
phenylethanol (1a) was used as the model substrate (see ESI).
1
5
activating motif compared to IL1 (Figure 1). These results
strongly prompted us to investigate the further design of ILꢀ
type activating agents for lipases by modifying the cationic
part. We were fascinated by the triazolium ILs because
various types of triazole derivatives have been prepared by the
1
6,17
Click chemistry,
and the triazolium cation has no acidic
proton on the triazolium ring. We hypothesized that the
triazoliumꢀtype alkyl PEG sulfate ILꢀcoated lipase PS may
influence the lipase activity in a different mode compared to
the imidazolium or phosphonium type IL when using them as
coating materials. We prepared three types of triazolium cetylꢀ
PEG10 sulfate (Tz1, Tz2, and Tz3) as model ILs and used
them as coating materials of lipase PS (Figure 1). The
resulting ILꢀcoated enzyme worked as an excellent catalyst of
the enantioselective transesterification. In particular, an
amazing improved stability of Tz1-PS was observed. We now
report the results of the investigation of the activating and
stabilizing properties of triazolium type alkylPEG sulfate
towards Burkholderia cepacia lipase.
Figure 2. IL-coated lipase PS-catalyzed enantioselective
transesterification.
It was found that Tz1-PS gave the best results for substrates 1d, 1g,
1
h, and 1i (Entries 4, 7, 8, and 9). However, IL1ꢀPS afforded the
best results for substrates 1a, 1b, 1c, 1e, 1f, and 1j. The Tz1ꢀPSꢀ
catalyzed reaction of naphthylalcohol (±)ꢀ1d proceeded 3,500 times
faster than that of the native lipase PS (Entry 4). It should also be
noted that Tz1-PS exhibited excellent results for pyrridyl alcohols
1
g, 1h, and 1i. The reaction rate of Tz1-PS was ca. twice those of
Figure 1. Design of ionic liquids as activating agents of lipase-
catalysed transesterification of secondary alcohols.
the IL1-PSꢀcatalyzed reactions for these substrates while
maintaining the excellent enantioselectivity (E >200). Tz2-PS and
Tz3-PS also gave good results for the reaction of (±)ꢀ1d and the
reaction rates were superior to that of IL1-PS (Entry 4). On the
other hand, there was a clear contrast for the reaction rate among
three triazolium saltsꢀcoated lipases when naphthylalcohol (±)ꢀ1e
was subjected to the reaction (Entry 5); Tz3-PS afforded the
highest reaction rate. On the contrary, that of Tz1-PS was the worst
among the ILꢀcoated enzymes, though the reaction rate was still
superior to that of the native PS. For the reaction of (±)ꢀ1k ,
unfortunately, all enzymes showed insufficient enantioselectivity,
though that of Tz1-PS reached to a practical level (Entry 11).
The results of the transesterification of methyl manderate ((±)ꢀ1l)
were confusing; Tz2-PS and Tz3-PS gave similar results with the
highest reaction rate, though the enantioselectivity slightly
decreased. These results clearly indicated that the substituent on the
triazolium ring strongly affects the enzyme substrate specificity.
Results and discussion
Transesterification of secondary alcohols using three types of
triazolium cetylPEG10 sulfate-coated lipase PSs.
18
We prepared three types of triazolium cetylꢀPEG10 sulfate salts
(
Tz1, Tz2, and Tz3), then used them as supporting materials
of lipase PS (for the details of synthesizing the triazolium ILs,
see the ESI). We evaluated these three types of triazolium ILꢀ
coated lipase PS by the enantioselective transesterification of
1
2 types of secondary alcohols (Figure 2) in diisopropyl ether
ꢀPr O) as the solvent in the presence of 1.5 equivalents of
vinyl acetate as an acyl donor at 35°C (Eq. 1 and Table 1). We
(
i
2
6
,7
also used commercial lipase PS and IL1-PS as the control
catalysts of the reactions. The results are dependent on both the
substrates and the coating ILs as shown in Table 1. The optimized
amount of the triazolium salt was 100 mole equivalent versus the
2
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Table 1. IL-supported lipase PS-catalyzed enantioselective transesterification.
ARTICLE
Entry Substrates Lipase PS
IL1-PS
a
Rate (E value)
Tz1-PS
a
Rate (E value)
Tz2-PS
a
Rate (E value)
Tz3-PS
a
Rate (E value)
a
Rate (E value)
b
b
b
b
b
1
2
3
4
5
6
7
8
9
1
1
(±)-1a
(±)-1b
(±)-1c
(±)-1d
(±)-1e
(±)-1f
(±)-1g
(±)-1h
(±)-1i
(±)-1j
90 (>200)
884 (>200)
1950 (>200)
569 (>200)
22 (>200)
14 (>200)
68 (147)
597 (>200)
582 (>200)
370 (>200)
35 (>200)
1.8 (>200)
67 (90)
631 (>200)
828 (>200)
338 (>200)
33 (>200)
8.8 (>200)
60 (105)
502 (>200)
984 (>200)
300 (>200)
27 (>200)
13 (>200)
63 (115)
62 (>200)
13 (>200)
0.01(>200)
0.01 (>200)
2.5 (>200)
11 (>200)
8.7 (>200)
6.9 (>200)
7.2 (>200)
6.6 (189)
531 (112)
341 (132)
383 (>200)
558 (>200)
295 (40)
940 (>200)
670 (>200)
570 (>200)
515 (>200)
168 (108)
592 (>200)
310 (>200)
65 (>200)
472 (>200)
160 (75)
606 (>200)
342 (>200)
145 (>200)
398 (>200)
219 (65)
0
1
1
7
(±)-1k
1
2
(±)-1l
-1
1.1 (180)
-1
12 (>200)
18 (151)
16 (142)
18 (140)
a
b
Rate: mM h , mg enzyme . Results using bold font showed the best ones among the tested enzymes. Calculated by % ee of (R)-
2
(ee
p
) and %
). see
ee of (S)-
1
(ee
s
). E= ln[(1-c(1+ee
p
))/ln[(1-c(1-ee
p
)), where c denotes conv. which was calculated by the following formula: c = ee
p
/(ee
p
+ee
s
ref. 12.
Aiming to obtain more insight into the IL mediated activation
of lipase PS, we next investigated the kinetic parameters for
these three immobilized enzymes using (R)ꢀ1ꢀ(pyridinꢀ3ꢀ
yl)ethanol (1h) as a model substrate, because Tz1-PS is
specially active for this compound (Table 2). As shown in this
table, the Km value of Tz1-PS is the lowest and this reflects
improvement in the K /K value; this indicated that coating
Table 2. Kinetic parameters for the lipase-catalyzed
transesterification of (R)-1-(pyridin-3-yl)ethanol (1h) using vinyl
acetate as an acyl donor
[
a]
Parameters
IL1-PS Tz1-PS
Tz2-PS
0.32
1.68
Tz3-PS
0.13
0.84
V
max
0.44
1.17
2.33
2.00
0.09
0.11
0.51
4.83
K
m
Kcat
cat/Km
1.65
0.70
cat
m
K
0.94
0.83
[a]
V
-1
-1
-1
-1
-1
with Tz1 allows an enhanced affinity of lipase PS towards this
max [M min mg(enzyme) ], K
m
[M], Kcat [min ], Kcat/K
m
[min M ]. Due to
the heterogenous reaction, the nonlinear least-squares method was
applied to the Michaelis-Menten type of equations: v = Vmax(E)mg[S ] / (K
[S ]), where Vmax is normalized by the weight of the lipase protein (E) mg.
The R values of all the experiments are in the range of 0.89 ~ 0.98; this
indicates that very reliable results have been obtained. see ESI.
alcohol. On the contrary, the K values of Tz2-PS and Tz3-
m
0
0
m
PS were 15ꢀfold and 8ꢀfold greater than that of Tz1-PS
,
+
0
respectively.
2
It is known that the lipase PS protein has a lid consisting of
hydrophobic amino acid chains that covers the entrance portion
of the enzyme. Colton, Kazlauskas and coꢀworkers revealed that
treatment of the Candida rugose lipase by an alcohol, such as iꢀ
PrOH through lyophilization altered the lid into an open form
Recyclable use of Tz1-PS and its stability in an IL solvent
One of the most important benefits of using the ILs is that it allows
the recyclable use of the enzyme if the reaction is possible in an
ionic liquid solvent. Thus, we next focused our attention on the
recyclable use of the enzyme in an IL solvent system using
Tz1-PS. To this end, we evaluated four types of ILs as solvents; i.
1
9
resulting in activation of the enzymatic reaction. Quilles et al.
reported that a similar activation phenomenon was observed
when the lipase from Thermomyces lanuginose or Candida
2
0
antarctica was treated with a surfactant. It was also reported
that water molecules significantly affected the activity of lipases in
8a
8b
8c
e., [P444ME][NTf
2
8c
],
[P444MEM][NTf
2
],
]. The Tz1-PSꢀcatalyzed reactions used four
substrates (Table 3). Among the solvents tested,
N] gave the best results for two substrates, (±)ꢀ
and (±)ꢀ1j (Entries 1 and 9 in Table 3). [P444MEM][Tf N]
was the suitable for indanol (±)ꢀ1b and [N221ME][Tf N] was
the best solvent for (±)ꢀ1k. Since [N221MEM][Tf N] afforded
[N221ME][NTf
2
],
and
21
the ILs. We assume that Tz1 may bind with a certain part of the
enzyme to produce the open form and activating it; the strong
affinity between the salt with the lipase protein might cause
[N221MEM][NTf
2
[N221MEM][Tf
2
4
preferable modification of the enzyme reactivity.
1
a
2
The importance of water in the activities of the enzymes in IL
2
22
solvents has been reported by several groups. There exist
numerous water molecules on the surface of the enzyme at the
2
good overall results, we chose this IL as the solvent for
demonstrating the recyclable use of Tz1-PS as illustrated in
Figure 3. The results are summarized in Table 4. Although
23
opposite part of the entrance of the active site of the enzyme. This
part was believed to be important for protein movement and
reported that maintaining the motion flexibility of the enzyme
protein might be important for displaying the enzyme activity.
Since the alkylꢀPEG moiety is essential regarding the increased
reaction rate, the alkylꢀPEG moiety in Tz1 may play an important
role in maintaining the flexibility of the enzyme.
Tz1-PS is still present in a suspended state in [N221MEM][Tf N] as
2
shown in Figure 3, no stripping of the ionic liquid coating
material (Tz1) from the enzyme into these IL solvents employed
during the reaction course was confirmed by an ion
chromatographic analysis (cation mode of ICꢀC4 column using a
5
% oxalic acid aqueous solution as the eluent).
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Table 3. Results of enantioselective transesterification of
secondary alcohols using Tz1-PS in an IL solvent system
.
Entry
Substrate
Solvent IL
Rate
E value
1
2
3
4
5
6
7
8
9
[N221MEM][Tf
[P444MEM][Tf
[N221ME][Tf
[P444ME][Tf
[N221MEM][Tf
[P444MEM][Tf
[N221ME][Tf
[P444ME][Tf
[N221MEM][Tf
[P444MEM][Tf
[N221ME][Tf
[P444ME][Tf
2
N]
1540
813
46
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
163
(±)-1a
2
N]
2
N]
2
N]
270
1818
1862
627
309
609
219
19
2
N]
(±)-1b
(±)-1j
2
N]
2
N]
2
N]
2
N]
1
1
1
0
1
2
2
N]
2
N]
2
N]
340
103
1
1
1
1
3
4
5
6
[N221MEM][Tf
[P444MEM][Tf
[N221ME][Tf
[P444ME][Tf
2
N]
114
112
135
67
38
23
40
32
(±)-1k
2
N]
2
N]
2
Figure 3. Recyclable system of Tz1-PS using [N221MEM][Tf N] as the
N]
solvent
2
-1
-1
Rate: mM h mg (enzyme) . The rate was determined by GC analysis after
a 30 min. reaction.
Table 4. Results of recyclable use of Tz1-PS in the
N] solvent system.
As expected, we succeeded in demonstrating the recyclable use of
Tz1ꢀPS when (±)ꢀ1a was subjected to the reaction along with an
excellent enantioselectivity; the initial reaction rate was maintained
after 10 repetitions of the reaction process (Entry 2, Table 4). We
usually removed the enzyme by filtration through a glass sintered
filter with a Celite pad and the IL was recovered after the
enzymatic reaction. However, we recognized that the Tz1-PS
[N221MEM][Tf
2
Entry
Run
Time
(R)-2a
(% ee)
>99
(S)-1a
(% ee)
92
Conv.%
b)
E
a
a)
b)
(h)
value
st
1
2
1
1
48
50
50
>200
>200
>200
th
10
2
>99
>99
>99
c)
3
After one
month
2
>99
d)
4
After 6
months
After 12
months
After 18
months
After 2
years
2
2
5
5
>99
>99
>99
98.5
>99
>99
79.1
>99
50
50
>200
>200
>200
>200
2
suspension in [N221MEM][Tf N] was left on a bench for a month at rt
d)
5
without this process. Thus, we investigated the activity of the
enzyme for the sample and found that the enzyme exhibited an
excellent activity the same as that of the freshly prepared one
d)
6
44.2
50
d)
7
(
Entry 3).
Inspired by these results, we prepared a native lipase PS
suspension in [N221MEM][Tf N] and stored it in a refrigerator
4°C), then checked its activity 3 months later compared the initial
a)
b)
Determined by HPLC (Chiralcel OB-H, n-hexane:2-PrOH= 20:1).
) and %ee of (S)-1 (ee ). E= ln[(1-
)), where c denotes the conv. which was calculated
2
Calculated by %ee of (R)-2 (ee
c(1+ee ))/ln[(1-c(1-ee
p
s
(
p
p
reaction rate of the transesterification of 1ꢀphenylethanol, because
it was difficult to evaluate the activity using the conventional
Lipase Kit due to the IL. We previously established that lipase PS
displayed an ca. 78% activity after 62 days at 25°C in 1ꢀbutylꢀ2,3ꢀ
c]
d]
by the following formula: c = ee
Stored at 4°C in a refrigerator.
p p s
/(ee +ee ). Stored at rt (ca. 25°C).
It was found that the stability of Tz1-PS was indeed superior to the
native PS and IL1-PS in this IL; the activity of IL1-PS slightly
dropped (ca. 80% of the initial one) after 6 months, and that of the
native PS was very low because no detectable amount of the
product was obtained by a 2ꢀhours reaction using the enzyme
stored for 6 months in the IL. On the other hand, to our surprize,
Tz1-PS displayed an excellent reactivity (Entry 4) and maintained
its initial activity even after 12 months (Entry 5). The reactivity of
Tz1-PS slightly dropped, but the enzyme still maintained an
excellent reactivity after 18 months (Entry 6), thus we finally found
that Tz1-PS, which was stored for 2 years in the IL, still displayed
an excellent reactivity (Entry 7), though it underwent an extraction
process 15 times during this testing. No study of the enzyme
2
4,25
dimethylimidazolium tetrafluoroborate ([bdmim][BF
expected, the native lipase PS maintained its activity more than
0% of the original one. It had thus been established that
4
]).
As
8
[
[
N221MEM][Tf N] could stabilize the lipase PS more than
bdmim][BF4]. Encouraged by the result, we decided to more
2
carefully investigate the stability of the native PS, IL1-PS and Tz1-
PS in [N221MEM][Tf N]
2
.
4
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ARTICLE
stability in an IL by investigating such a long time has been
reported to date.
was also confirmed that glycine did not influence the activity but only
1
4
contributed to the stabilization. We also confirmed that the reactivity
of the Celite free Lipase PS obtained by the lyophilization of the
supernatant was similar to that of commercial Lipase PS (see ESI).
Lipase powder, which was immobilized with the appropriate solid
materials is generally stable, however, they quickly lost their
activity once dissolved in a solvent in the absence of a substrate. It
was really an amazing fact that Tz1-PS possesses a high activity
after a 2-year storage in an IL. The tested sample was only 5.0 mg
7
a,14
However, the stability was significantly decreased.
now commercially available from Tokyo Chemical Industry Co., Ltd.
TCIꢀB3028).
The IL1-PS is
(
2
of Tz1-PS in 1.0 mL of [N221MEM][Tf N] (Figure 3). In addition,
the enzyme underwent the extraction process many times, which
might be very harsh conditions for the protein. We assume that the
cooperative effect of the IL, [N221MEM][Tf N] and Tz1 coating
2
Preparation of Tz1-PS.
To an acetone (2 ml) solution of 1ꢀbutylꢀ3ꢀmethylꢀ1,2,3ꢀ
triazolium iodide (0.52 g, 1.87 mmol) was added ammonium
cetylꢀPEG10 sulfate (1.46 g, 1.87 mmol), and the mixture was
stirred at rt for 24 h, then diluted with acetone and cooled to ꢀ
might allow this amazing stabilization of the enzyme.
2
5°C to form ammonium iodide as a white precipitate which
Conclusions
was removed by filtration through a membrane filter, then the
filtrate was treated with active charcoal (2.12 g) for 6 h at
3
sintered filter with a Celite pad to remove the active charcoal,
then the filtrate was filtratered through an alumina column
In conclusion, three types of triazolium cetylꢀPEG10 sulfate ionic
liquids were synthesized and their property for activation of
Burkholderia cepacia lipase evaluated; the resulting ILꢀcoated
enzymes well catalyzed the enantioselective transesterification of
various secondary alcohols using vinyl acetate as an acyl donor.
However, both the reaction rate and enantioselectivity depended on
the substituent on the triazolium ring of the ILs. Among the tested
ILꢀcoated enzymes, 1ꢀbutylꢀ3ꢀmethylꢀ1,2,3ꢀtriazolium cetylꢀ
PEG10 sulfate (Tz1)ꢀcoated lipase PS (TZ1-PS) was especially
suitable for the transesterification of 1ꢀ(pyridinꢀ2ꢀyl)ethanol, 1ꢀ
5°C. The resulting mixture was filtered through a glass
(5.25 g, Type III activated), and lyophilized to give 1ꢀbutylꢀ3ꢀ
methylꢀ1,2,3ꢀtriazolium cetylꢀPEG10 sulfate (Tz1) (0.497 g,
1
0
.55 mmol) in 29% yield: mp 28.3 °C; H NMR (600 MHz,
CDCl = Hz) δ 0.88 (3H, t, = 7.2), 1.00 (3H, t, = 7.2), 1.25
30H, m), 1.46 (2H, sextet, = 7.2), 1.60 (2H, m), 3.4 (2H, t,
= 5.4), 3.65ꢀ3.63 (34H, m), 3.72 (2H, m), 4.25
3
,
J
J
J
(
J
J=
6
.6), 3.58 (2H, d, J
(
pyridinꢀ3ꢀyl)ethanol, and 1ꢀ(pyridinꢀ4ꢀyl)ethanol. Furthermore,
Tz1-PS exhibited almost the same activity as that of the freshlyꢀ
prepared one even after storage for 2 years in [N221MEM][Tf N]. The
13
(3H, s), 4.73 (2H, t, = 7.8), 9.22 (1H, s), 9.44 (1H, s)
J
;
C NMR
(150 MHz, CDCl , J=Hz) δ 13.36, 14.09, 22.64, 25.76, 26.04,
3
2
2
4
7
9.31, 29.45, 29.49, 29.52, 29.57, 29.60, 29.64, 31.41, 31.87,
1.12, 54.08, 61.49, 61.67, 61.70, 62.79, 69.98, 70.10, 70.13,
0.25, 70.42, 71.50, 71.71, 72.50, 72.66, 131.41, 132.29; IR (neat,
results indicate the possibility of the IL engineering for enzyme
stabilization. Further investigations about the origin of the
stabilizing effect of the IL coating for enzymes are now in progress
in our laboratory.
ꢀ1
cm ) 3421, 2916, 2850, 1538, 1466, 1345, 1280, 1242, 1109, 964,
+
8
1
42; ESIꢀMS (Cation): calcd for C H N 140.1182 , found
7 14 3
40.1180.
ꢀ
4
Commercial lipase PS (1.00 g, enzyme protein 10 mg; 3.1 x 10
mmol) was dissolved in 10 ml of 0.1 M phosphate buffer (pH 7.2)
and the mixture was centrifuged twice at 3,500 rpm for 5 min, then
Experimental
General experimental method for synthesis of triazolium salt ILs
ꢀ2
the resulting supernatant was mixed with Tz1 (28.2 mg, 3.1 x 10
mmol). The mixture was stirred for 15 min at rt, then lyophilized to
afford Tz1-PS (262 mg). Because the enzyme protein and Tz1
might not be removed during the process, we estimated the enzyme
content in this Tz1-PS powder was 3.8 % (w/w) and the remaining
was 11% (w/w) Tz1 and very fine inorganic materials derived from
the Celite or potassium phosphate buffer. Since the commercial
The ILs were prepared according to previously reported
1
13
procedures. The H and C NMR spectra were recorded by a
1
13
Bruker Avance III (600 MHz for H and 150 MHz for C).
High resolution mass spectra were recorded by a Thermo
Fisher
Scientific
EXACTIVE
mass
spectrometer.
Abbreviations of thesolvent ILs are as follows: tributyl((2ꢀ
ꢀ4
lipase PS included 1.0 % lipase protein (3.1 x 10 mmol), 20 wt %
glycine, and the rest Celite. The 10 ml of the 0.1 M potassium
methoxyethyl)phosphonium
bis(trifluoromethanesulfonyl)amide
tributyl((2ꢀ
8
a
2
([P444ME][Tf N]) ,
2 4
phosphate buffer included 124.9 mg of K HPO and 38.5 mg of
methoxyethoxy)methyl)phosphonium
bis(trifluoromethanesulfonyl)amide ([P444MEM][Tf
8
b
KH PO , thus the theoretical amount of TZ1-PS after lyophilization
2
4
2
N]) ,
N,Nꢀ
was calculated to be 401.6 mg. However, the amount of the obtained
powder was 262 mg. We repeated this several times, and similar results
were obtained. We suppose that the glycine and K HPO or KH PO
2 4 2 4
diethylꢀ2ꢀmethoxyꢀ
bis(trifluoromethanesulfonyl)amide ([N221ME][Tf
ethylꢀ ꢀ((2ꢀmethoxyethoxy)methyl)ꢀ
bis(trifluoromethanesulfonyl)amide ([N221MEM][Tf
Nꢀmethylethanaminium
8
c
2
N]),
and N-
N
Nꢀmethylethanaminium
form complexes, the some of which were removed by Celite absorption
during the process. Since Khmelnitsky et al. reported the potassium
8c
2
N]) . Lipase PS
was provided by Amano Enzyme Co., Ltd. The protein content of the
commercial lipase PS is cited as 1.0 wt% and the remainder is 20 wt%
glycine as a stabilizing agent and Celite as a supporting material. The
enzyme is a single protein and its amino acid sequence is known and
the molecular weight can be precisely determined as 33,295 Dalton. It
26
saltꢀcoated activation of lipase, we carefully investigated the activity
of the lipase in the presence or absence of TZ1. It was confirmed that
TZ1 was essential for producing a significant activation of the enzyme
(see ESI).
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Tz2-PS and Tz3-PS were prepared by a similar method and
contained enzyme proteins 4.0 %(w/w) and 3.6 %(w/w),
respectively. Since the ILꢀcoated enzymes are very hygroscopic,
these should be stored in a desiccator.
Determination of kinetic parameters for the reaction of (R)-1h.
The reaction mixture was sampled at the appropriate reaction
interval (10 min, 15 min, and 20 mim) and determined the %
conversion by capillary GC analysis (Quadrex bonded fused silica
methyl silicone,
internal reference. We conducted reactions at various enzyme
concentration conditions, Vmax, K , and Kcat values were thus
φ 0.25 mm × 25 m, He) in the presence of an
Typical enzymatic reaction.
The reaction was typically carried out as follows: To a mixture of
m
5.0 mg of Tz1-PS (0.19 mg of the enzyme protein) in 1.0 mL of
obtained. The reproducibility of the measurement technique was
solvent ( ꢀPr O or IL) was added (±)ꢀ1a (50 mg, 0.41 mmol) and 55
i
2
2
confirmed by the sufficiently high R value. See Figures S1~ S4 in
mg of vinyl acetate (1.5 equiv.) and the resulting mixture was
stirred at 35 °C. To evaluate the initial reaction rate, the reaction
was conducted in the presence of 0.5 mmol of hexadecane as an
internal reference, an aliquot of the reaction mixture was sampled
at 30 min. reaction and extracted with a mixed solvent of diethyl
ether and hexane (1:4), and the rate was determined by capillary
GC analysis. The reaction course was monitored by silica gel thin
ESI.
Acknowledgements
The present study was supported by a JSPS KAKENHI
KIBAN(A) Grant (No.26241030).
layer chromatography (TLC) and the product (
alcohol ( )ꢀ1a were extracted with a mixed solvent of diethyl ether
R)ꢀ2a and unreacted
S
Notes and references
and hexane (1:4) when the spots became the same size, then
purified by silica gel TLC. Since it is well recognized that the water
content of the solvent influences the lipase performance, the ionic
liquids were dried under reduced pressure at 50°C at 1.0 Torr for 3
to 5 hours prior to the reaction. The enantiomeric excess of the
product acetate and unreacted alcohol were determined by HPLC
1
.
Reviews see. (a) C.ꢀH. Wong, G. M. Whitesides, Enzymes in
Synthetic Organic Chemistry, Pergamon, Oxford, (1994). (b)
M. Klivanov, Trends Biotechnol. 1997, 15, 97ꢀ101. (c) U. T.
Bornscheuer, R. J. Kazlauskas, Hydrolases in Organic
Synthesis: Regioꢀand Stereoselective Biotransformations, John
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& Sons Inc., Chichester (1999). (d) K. Faber,
(
Chiralcel OBꢀH, nꢀhexane: 2ꢀpropanol= 9:1 or 20:1). The reaction
th
Biotransformations in Organic Chemistry, A Textbook, 6
Edition. Springer, Heidelberg Dordrecht London New York,
(2011).
rate was determined by GC analysis after 30 min. reaction in the
presence of an internal reference. The enantioselectivity of the
8
reaction was displayed as the E value which was calculated by
2. Reviews see: (a) P. Wasserscheid, T. Welton (eds.)."Ionic
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%
ee of (
c(1ꢀee )); where, c denotes the conv. which was calculated by the
p s
following formula according to the reference : c = ee /(ee +ee ).
p s p
R)ꢀ2 (ee ) and %ee of (S)ꢀ1 (ee ). E= ln[(1ꢀc(1+ee ))/ln[(1ꢀ
p
8
p
2
363ꢀ2389; (d) J. Ranke, S. Stolte, R. Störmann, J. Arming, B.
The water contents of our tested solvents (diisopropyl ether and
ILs) were maintained at less than 400 ~ 500 ppm. This water
content had no influence on the reaction, because it was reported
that more water (at least 1.25 %) was needed to affect the lipase
Jastorff, Chem. Rev. 2007, 107, 2183ꢀ2206; (e) N. V.
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State of the Art, Intech, Croatia, (2015).
.
2
7
activity.
Typical reaction conditions for recyclable use of Tz1-PS in ionic
liquid solvent system.
3. Typical reviews of enzymatic reactions using ILs as solvents:
(a) R. A. Sheldon, R. M. Lau, M. J. Sorgedrager, F. van
Rantwijk, K. R. Seddon, Green Chem. 2002, 4, 147ꢀ151. (b) U.
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equiv.) in [N221MEM][Tf N] (1.0 mL) was added Tz1-PS (5.0 mg,
2
corresponding to 0.19 mg of enzyme) and the mixture was stirred at
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isolated from the ether layer. It was essential to repeat the
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2
011, 89, 493ꢀ499. (h) Ed. Pablo Domínguez de María, Ionic
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R)ꢀ2a and
(S)ꢀ1a were separated by preparative silica gel TLC. Since the
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repeatedly use the lipase (see Figure 3); the ionic layer was placed
under reduced pressure (1 Torr) at room temperature for 15 min. to
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set of substrate and acyl donor ((±)ꢀ1a (50 mg) and vinyl acetate
(
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6
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