Increased enantioselectivity and remarkable acceleration of lipase-catalyzed transesterification by using an imidazolium PEG-Alkyl sulfate ionic liquid

Article abstract of DOI:10.1002/chem.200601043

Several types of imidazolium salt ionic liquids were prepared derived from poly(oxyethylene)alkyl sulfate and used as an additive or coating material for lipase-catalyzed transesterification in an organic solvent. A remarkably increased enantioselectivity was obtained when the salt was added at 3-10 mol % versus substrate in the Burkholderia cepacia lipase (lipase PS-C)-catalyzed transesterification of 1-phe nylethanol by using vinyl acetate in diisopropyl ether or a hexane solvent system. In particular, a remarkable acceleration was accomplished by the ionic liquid coating with lipase PS in an iPr₂O solvent system while maintaining excellent enantioselectivity; it reached approximately 500- to 1000-fold acceleration for some substrates with excellent enantioselectivity. A similar acceleration was also observed for IL1-coated Candida rugosa lipase. MALDITOF mass spectrometry experiments of the ionic-liquid-coated lipase PS suggest that ionic liquid binds with lipase protein.

Full text of DOI:10.1002/chem.200601043

DOI: 10.1002/chem.200601043
Increased Enantioselectivity and Remarkable Acceleration of Lipase-
Catalyzed Transesterification by Using an Imidazolium PEG–Alkyl Sulfate
Ionic Liquid
Toshiyuki Itoh,*[a] Yuichi Matsushita,[a] Yoshikazu Abe,[a] Shi-hui Han,[a] Shohei Wada,[a]
Shuichi Hayase,[a] Motoi Kawatsura,[a] Shigeomi Takai,[a] Minoru Morimoto,[b] and
Yoshihiko Hirose[c]
Abstract: Several types of imidazolium
salt ionic liquids were prepared derived
from poly(oxyethylene)alkyl sulfate
and used as an additive or coating ma-
terial for lipase-catalyzed transesterifi-
cation in an organic solvent. A remark-
ably increased enantioselectivity was
obtained when the salt was added at 3–
10 mol% versus substrate in the Burk-
nylethanol by using vinyl acetate in di-
isopropyl ether or a hexane solvent
system. In particular, a remarkable ac-
celeration was accomplished by the
ionic liquid coating with lipase PS in an
iPr2O solvent system while maintaining
excellent enantioselectivity;it reached
approximately 500- to 1000-fold accel-
eration for some substrates with excel-
lent enantioselectivity. A similar accel-
eration was also observed for IL1-
coated Candida rugosa lipase. MALDI-
TOF mass spectrometry experiments of
the ionic-liquid-coated lipase PS sug-
gest that ionic liquid binds with lipase
protein.
AHCTREUNG
Keywords: acceleration · enantiose-
lectivity · ionic liquids · lipids ·
transesterification
A
catalyzed transesterification of 1-phe-
Introduction
enantioselectivity was recorded when (Æ)-1-phenylethanol
(1a) was acetylated by lipase PS-C in toluene,[4] while signif-
icant reduction in enantioselectivity was obtained when the
reaction was carried out in diisopropyl ether (iPr2O) as sol-
vent. Therefore, development of a strategy to improve
lipase reaction performance in an organic solvent system is
desirable.[1,3]
Several methods have been reported for activation of li-
pases in a nonaqueous medium: lipid-coating-mediated acti-
vation by Okahata,[5] entrapment of lipases in hydrophobic
sol–gel materials by Reetz,[6] molecular bioimprinting by
Braco,[7] and salt-mediated activation by Dordick.[8] Among
these, the salt-mediated activation of lipases by the Dordick
group is quite interesting;[8] they indicated that enzymes
could be activated by a large amount of salts with kosmo-
tropic anions during the lyophilization process.[8] Recently,
Kim and Lee reported another very interesting salt-mediat-
ed activation of a lipase: Lipase PS was mixed with the ionic
liquid and resulted in “ionic-liquid-coated lipase PS”, which
showed a more increased enantioselectivity than that of
commercial lipase PS-C in toluene, though no significant
modification of the reaction rate was obtained.[9]
The value of an enzymatic reaction in organic synthesis is
greatly increased by its environmentally friendly aspect.[1]
Lipase PS from Burkholderia cepacia[2] is one of the most
widely used enzymes applicable for various substrates;[1]
however, the activity in nonaqueous media is reduced and it
has been reported that the enantioselectivity is significantly
dependent on the solvent system.[3] As an example, excellent
[a] Prof. Dr. T. Itoh, Y. Matsushita, Y. Abe, S.-h. Han, S. Wada,
Dr. S. Hayase, Dr. M. Kawatsura, Dr. S. Takai
Department of Materials Sciences
Faculty of Engineering, Tottori University
4–101 Koyama-minami Tottori 680–8552 (Japan)
Fax : (+81)857-31-5259
E-mail: titoh@chem.tottori-u.ac.jp
[b] Dr. M. Morimoto
Research Centre for Bioscience and Technology
Tottori University, 4–101 Koyama-minami
Tottori 680–8552 (Japan)
[c] Dr. Y. Hirose
Amano Enzyme Ltd., Kagamihara
Gifu 509–0108 (Japan)
Room-temperature ionic liquids are a new class of sol-
vents and have attracted growing interest recently because
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
of their unique physical and chemical properties;they are
nonvolatile, nonflammable, have low toxicity, and good solu-
bility for many organic and inorganic materials.[10] We, in the
Bioscience and Technology group, established that lipase-
catalyzed transesterification could be conducted in an ionic-
liquid solvent system;[11,12] the enantioselectivity was depen-
dent on the anionic part of the imidazolium salts.[10–13] It has
been reported that some surfactants were able to stabilize
enzymes in an ionic liquid solvent system;the groups of
Russell[14] and Goto[15] independently reported that PEG
treatment significantly stabilized the enzyme reactivity in an
ionic-liquid solvent system, and made it possible to modify
the enantioselectivity. Lipase reactivity in an organic solvent
system can be modified by several reported compounds:
crown ethers,[16,17] cyclodextrins,[18] surfactants,[7,19] and a polar
solvent such as dimethylsulfoxide (DMSO),[20] or even water.[21]
Based on these results, we investigated the possibility of
regulating the enantioselectivity and reactivity of lipase PS
by using iPr2O as the solvent. We expected that optimization
of an appropriate combination of the anionic part and the
imidazolium cation of the ionic
to improve enzyme enantioselectivity in a nonaqueous sol-
vent system, the simplest being the addition of a suitable
compound that affects the enzyme activity. We previously
reported that increased enantioselectivity occurred when the
reaction was carried out in the presence of a trace amount
of thiocrown ether.[16] Ueji et al. reported that enantioselec-
tivity of Candida rugosa lipase (CRL) was increased by the
addition of DMSO as cosolvent in toluene.[20] Modified
enantioselectivity was also reported if the reaction was car-
ried out in an ionic-liquid solvent instead of a usual organic
solvent.[9–13,22] We therefore investigated the lipase-catalyzed
acylation of (Æ)-1-phenylethanol (1a) in the presence of
various additives, in particular, ionic-liquid additives by
using iPr2O as solvent (Table 1).
The enantioselectivity of lipase PS-C for (Æ)-1a is signifi-
cantly dependent on the reaction medium and the E
value[22] is just 16 for iPr2O (entry 1), while it is over 200 for
toluene (entry 2). We found that a remarkably increased
enantioselectivity was obtained when the reaction was car-
ried out in the presence of 10 mol% (versus substrate) of
liquids might make it possible
Table 1. Results of the additive effect on the lipase PS-catalyzed enantioselective acylation of (Æ)-1-phenyl-
ethanol (1a)
to design a useful regulator of
the lipase-catalyzed reaction.
We prepared several types of
imidazolium poly(oxyethylene)
alkyl sulfate ionic liquids and
used them as an additive or
coating material for lipase PS;
a remarkably increased enan-
tioselectivity was obtained
when the ionic liquid was
added at 3 mol% versus the
substrate in the lipase PS-C-
catalyzed transesterification of
(Æ)-1a by using vinyl acetate
as acyl donor in an iPr2O sol-
vent system. Further, we dis-
covered that an extraordinary
acceleration was accomplished
by our ionic-liquid coating
with lipase PS in an iPr2O sol-
vent system, while maintaining
AHCTREUNG
Solvent Enzyme additive[a]
iPr2O PS-C-none
toluene PS-C-none
t[h] Yield[b] [%] of
Yield[b] [%] of
Conversion E value[d]
(R)-2a (eep, [%])[c] (S)-3a (ees, [%])[c] [%][d]
1
2
3
4
5
6
7
8
9
28
28
28
28
24
21
(83)
31
61 (38)
40 (76)
58 (66)
50 (71)
64 (20)
55 (24)
67 (35)
66 (33)
50 (>99)
57 (35)
72 (15)
61 (41)
32
43
40
41
31
20
27
26
50
32
15
29
16
>200
>200
>200
3
(99)
34
(>99)
33
(>99)
15
(43)
16
(98)
19
(95)
15
(92)
33
(>99)
30
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
PS-C+[14]-ane-S4
PS-C+DMSO
PS-C+[bmim][BF4]
T
PS-C+[bdmim]BF4] 24
125
PS-C+[bdmim]
PS-C+Brij56[e]
PS-C+IL1
A
55
excellent
enantioselectivity,
and even better enantioselec-
tivity was obtained for some
substrates.[12] Herein, we wish
to report the details of our
method of ionic-liquid-promot-
ed activation of the lipase.
26
28
29
26
24
35
>200
10
10 iPr2O
11 iPr2O
12 iPr2O
PS-C+IL2
(70)
9
(87)
24
CF-PS[f]-none
CF-PS[f] +IL1
17
>200
(>99)
Results and Discussion
[a] 10 mol% versus (Æ)-1a. [b] Isolated yield. [c] Determined by HPLC (Chiralcel OB, hexane/iPrOH=8:1).
[d] Calculated from eep and ees. E=ln (1+eep)]/ln[(1Àc)(1Àeep)], in which c is conversion, which was cal-
[(1Àc)
culated by the following formula: c=eep/(eep+ees). See ref. [23]. [e] Brij56=poly[oxyethylene(10)] cetyl ether
(C16H33(OCH2CH2)nOH, n~10). [f] The reaction was carried out in the presence of 0.5 wt% (versus substrate)
of CF-PS.
A
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Additive effect on the lipase-
catalyzed transesterification:
Several methods are reported
AHCTREUNG
AHCTREUNG
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9229

T. Itoh et al.
thiocrown ether, 1,4,8,11-tetrathiatetracyclodecane ([14]ane-
S4),[16] or DMSO (entries 3 and 4).[20] It was reported that
modified stereoselectivity sometimes occurred when the
lipase-catalyzed reaction was conducted in an ionic-liquid
solvent system,[9–13,22] and it was established that the lipase
activity was dependent on the combination of the cationic
and anionic parts of the imidazolium salts, in particular, the
anionic part of the ionic liquids.[14,24] It is also known that
some surfactants, such as poly(oxyethylene)alkyl ether,
affect lipase reactivity,[14,15] so we next tested the lipase PS-
C-catalyzed acetylation of (Æ)-1a in the presence of a sur-
factant, poly[ethylenoxy(10)] cetyl ether (Brij56)[25] or ionic
liquids. Interestingly, increased enantioselectivity was re-
corded for 1-butyl-2,3-dimethylimidazolium tetrafluorobo-
(entry 10). We discovered that ionic liquid IL1 worked as an
excellent additive to increase the enantioselectivity, and a
high E value was recorded even when IL1 was used as addi-
tive in the Celite-free PS (CF-PS)-catalyzed reaction
(entry 12).[26] This additional effect was found to be inde-
pendent of the presence of Celite as supporting material of
the lipase (entries 11 and 12).
Activation of the lipase by the ionic-liquid coating: Dordick,
Clark, and co-workers reported that lyophilization was es-
sential to activate the lipase by inorganic salt-mediated acti-
vation.[8] Recently, Goto et al. reported that a marked accel-
eration was obtained for PEG-supported lipase PS, which
was prepared by lyophilization of an enzymatic buffer solu-
tion in the presence of PEG, in an organic solvent system.[15]
However, Kim and Lee prepared their “ionic-liquid-coated
lipase” by just mixing the lipase with a melted ionic liquid.[9]
Further, Braco established that lyophilization was essential
to achieve activation of the enzyme by a bioimprinting pro-
tocol.[7] From these results, we hypothesized that the coating
method might be the key point for activation of lipases by
an ionic liquid;lyophilization of an enzyme in the presence
of the ionic liquid might be essential to activate the lipase.
We thus prepared “IL1-coated PS” by the following
method: Commercial lipase PS-C (1.0 g, involving approxi-
mately 1 wt% of the enzyme protein) was added to potassi-
um phosphate buffer (10 mL, 0.1m, pH 7.2);this mixture
was stirred for 5 min at room temperature, then centrifuged
at 3500 rpm for 5 min. The resulting supernatant was mixed
with IL1 and the solution was lyophilized to give IL1-PS
(250 mg).[27] Celite-free lipase PS (CF-PS) was also prepared
by lyophilization of the supernatant of the mixture of com-
mercial lipase PS-C in 0.1m phosphate buffer (pH 7.2) after
centrifugation.[27] The transesterification of (Æ)-1a by using
these ionic-liquid-coated enzymes was extremely successful
and the results are summarized in Table 2.
A remarkable acceleration was accomplished by using
IL1-PS whilst maintaining excellent enantioselectivity. Re-
action conversion reached 44% after just 1.3 h when IL1-
PS, which was prepared by using about 50 equivalents of
IL1 versus lipase protein, was used as catalyst in iPr2O
(entry 1), while it took 28 h at only 32% conversion when
commercial PS-C was used as catalyst, as mentioned earlier
(Table 1, entry 1). The results of the activation of lipase PS
depended on the amount of IL1 in the coating process;the
best result was recorded when IL1-PS was prepared by
using approximately 100 equivalents of IL1 versus lipase
protein (entry 2). The use of a large amount of IL1 (about
300 or 500 equivalents versus lipase protein) caused a slight
decrease of both enantioselectivity and reaction rate (en-
tries 3 and 4). Enantioselectivity significantly depends on
the cationic part of the ionic liquids: no marked increased
enantioselectivity was recorded for IL2-PS (entry 5) and
only a slight increase was obtained for Salt1-PS (entry 7). A
remarkable acceleration was again obtained for Brij56-PS,
while this enzyme showed only slightly increased enantiose-
lectivity (entry 8). These results seem to suggest that the
rate ([bdmim]
azolium hexafluorophosphate [bdmim]
a significant decrease was obtained by addition of 1-butyl-2-
methylimidazolium tetrafluoroborate [bmim][BF4] (entry 5).
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
Further, a slight increase of enantioselectivity was recog-
nized when Brij56 was added to the reaction mixture
(entry 8). These results prompted us to prepare a hybrid of
Brij56 with an imidazolium salt, and to evaluate its effect on
the lipase activity.
Two types of imidazolium salts, [bdmim][cetyl-PEG10-sul-
fate] (IL1) and [bmim][cetyl-PEG10-sulfate] (IL2) were
synthesized:[12] A mixture of Brij56 with ammonium amido-
sulfate was stirred for 17 h at 1108C and dried under re-
duced pressure at 66.7 Pa and at 608C for 3 h to give ammo-
nium Brij56 sulfate as white powder. This was treated with
an equivalent amount of imidazolium chloride in acetone
with vigorous stirring at room temperature for 6 h to form
ammonium chloride as a white precipitate, which was re-
moved by filtration through a glass-sintered filter with a
Celite pad. The resulting filtrate was evaporated and the
residue was again dissolved in acetone and filtered through
an alumina (neutral type I, activated) short column. The fil-
trate was evaporated and dried under reduced pressure to
form IL1 and IL2 with 70–74% overall yield, respectively.
The salts showed melting points at 35–378C due to their am-
phiphilic nature and they dissolved easily in many types of
organic solvents and in water.
There was a clear contrast in the modification effect be-
tween these two ionic liquids on the lipase PS-C-catalyzed
reaction (entries 9 and 10);a remarkably increased enantio-
selectivity was accomplished when the reaction was carried
out in the presence of approximately 10 mol% (versus sub-
strate) of IL1, which had a methyl group at the 2-position
on the imidazolium ring, while no marked change of enan-
tioselectivity was recorded when IL2, which has a proton at
the 2-position on the imidazolium ring, was used as additive
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Ionic Liquids
FULL PAPER
Table 2. Results of additive effect on the lipase PS-catalyzed enantioselective acylation of (Æ)-1-phenyletha-
nol (1a).
pared to that of IL1-PS
(entry 2). This was assumed to
be because lipase activity was
rapidly lost during the lyophili-
zation process without glycine.
Since it was anticipated that
the alkyl sulfate anion might
have an impact on the lipase
reactivity, we next attempted
to evaluate the poly(oxyethyl-
CM[a]
Molar ratio
enzyme:CM
t[h]
Yield[b] [%] of
Yield[b] [%] of
Conversion
[%][d]
E value[d]
(R)-2a (eep, [%])[c]
(S)-3a (ees, [%])[c]
A
using (Æ)-1b[16c,d] as a model
substrate;the reaction of ( Æ)-
1b proceeds very rapidly,
making it possible to deter-
mine the reliability of the ki-
netic parameters, although
only a small modification of
1
2
3
4
5
6
7
8
9
IL1
IL1
IL1
IL1
1: 50
1:100
1:300
1:500
1:100
1:100
1:100
1:300
1.3
1.0
2.5
3.5
2.0
2.0
1.0
24.0
48.0
24.0
30 (>99)
42 (>99)
25 (>99)
27 (>99)
36 (67)
33 (94)
27 (84)
30 (>99)
21 (99)
27 (98)
46 (79)
42 (98)
66 (36)
52 (58)
43 (88)
47 (79)
77 (58)
46 (32)
60 (47)
56 (78)
44
50
27
37
57
45
48
24
32
43
>200 (470)
>200 (1060)
>200 (285)
>200 (360)
14
IL2
Salt1[e]
Brij56
IL1[f]
IL1[g]
IL1[g]
75
27
>200 (270)
>200 (315)
>200 (217)
1:100
1:100+Gly[h]
the
enantioselectivity
was
10
found for the 1L1-PS-catalyzed
acetylation of (Æ)-1b. Ten
types of ionic-liquid-coated li-
pase PS and Brij56-PS were
prepared and used as catalysts
in the transesterification of
(Æ)-1b (Table 3). We discov-
[a] CM=coating material. [b] Isolated yield. [c] Determined by HPLC (Chiralcel OB, hexane/iPrOH=8:1).
[d] Calculated by eep and ees. E=ln (1+eep)]/ln[(1Àc)(1Àeep)], in which c is conversion, which was calcu-
[(1Àc)
lated by the following formula: c=eep/(eep+ees). [e] Salt1: [NH4][C16H33(OCH2CH2)10OSO3]. [f] CF-PS was
mixed with IL1 melted by warming and used immediately for the reaction. [g] IL1 coating lipase was prepared
by using a pure lipase protein. [h] Lyophilization was carried out in the presence of 100 equiv of IL1 and gly-
cine (versus lipase protein).
A
G
E
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G
U
ACHTREUNG
present activation might be caused by the result of an inter-
action of a poly(oxyethylene)alkyl group and a cationic part
of the ionic liquid. It was confirmed that lyophilization of an
enzyme in the presence of the ionic liquid was essential to
activate the lipase because no significant acceleration was
recorded when CF-PS was mixed with IL1 that had been
melted by warming, and the resulting enzyme was used im-
mediately for acylation (entry 8). However, increased enan-
tioselectivity was obtained in this case too (entry 8). These
results seem to suggest that there might be a different origin
for the increased enantioselectivity and acceleration.
ered that IL1 was the best ionic liquid to activate lipase PS
(entry 8). The reaction rate was drastically accelerated and
reached 98-fold acceleration over commercial lipase PS-C
when IL1-supported lipase PS was used as catalyst (entry 8).
The second choice was IL1c-PS with which 90-fold accelera-
Table 3. Results of ionic-liquid-coated lipase PS-catalyzed enantioselec-
tive acylation of (Æ)-3-hydroxypentanenitrile (1b).
Table 2 shows the results of ionic-liquid-coated lipase PS-
catalyzed enantioselective acylation of (Æ)-1-phenylethanol
(1a). During these experiments, we found that the amount
of IL1-PS was about 200–300 mg per 1.00 g of PS-C after
lyophilization even though it was dried very carefully. Since
it was reported that commercial PS-C was prepared by sup-
porting 1.0 wt% of pure enzyme protein with Celite, the
amount of IL1-PS was evidently too large based on the in-
formation of PS-C provided from Amano Enzyme Ltd: We
used only 29 mg of IL1 per 1.00 g of PS-C. The question
was resolved by Amano Enzyme Ltd;commercial PS-C con-
tains 20 wt% of glycine that was used as essential stabilizer
during the preparation of PS-C by the lyophilization process.
So, we prepared IL1-coated PS by using pure enzyme pro-
tein that was provided by Amano Enzyme Ltd and tested its
activity;no acceleration was obtained though excellent
enantioselectivity was still recorded (entry 9). We next pre-
pared IL1-pure PS by lyophilization in the presence of
100 equivalents of glycine. An increased reaction rate was
indeed obtained (entry 10), but it was not significant com-
Coating material[a]
PS-C
Rate
Specific
activity
[mhÀ1 mgÀ1
]
1
2
3
4
5
6
7
8
9
0.10
0.85
3.9
6.4
7.9
4.1
6.7
9.8
9.0
8.1
6.4
6.8
1
9
A
ACHTREUNG
A
N
39
64
79
41
70
98
90
81
64
68
ACHTREUNG
IL1a (from Brij35)
IL1b (from Brij52)
IL1 (from Brij56)
IL1c (from Brij58)
IL1d (from Brij92)
IL1e (from Brij97)
IL1 f (from Brij700)
10
11
12
[a] IL1=[bdmim]
[C16H33
N
[C12H25-
E
A
A
ACHTREUNG
[bdmim][C16H33(OCH2CH2)20OSO3]; IL1d=[bdmim][C18H35(OCH2CH2)2-
OSO3]; IL1e=[bdmim][C18H35(OCH2CH2)10OSO3]; IL1 f=[bdmim][C18H35-
(OCH2CH2)100OSO3].
G
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T. Itoh et al.
Table 4. Results of the activation effect by the IL1 coating on the li-
tion was recorded (entry 9). It is interesting that [bdmim]-
[BF4] gave better results than [bmim][BF4] (entries 2 and 3).
pase PS-catalyzed enantioselective acylation of (Æ)-1.
G
ACHTREUNG
It was thus confirmed that the cationic group of the ionic
liquid played an important role in the increased enantiose-
lectivity of lipase PS.
We further discovered a very interesting activation prop-
erty of IL1-PS: the acceleration of the rate was significantly
dependent on the substrates (Table 4). A truly remarkable
acceleration was accomplished for 1a, 1b, 1d,[29] 1e,[30] 1h,[33]
1k,[36] and 1i;[35] 1100-fold acceleration was obtained for al-
cohol, (Æ)-1d, 500-fold for (Æ)-1e, 98-fold for (Æ)-1b, 26-
fold for (Æ)-1l,[11f] and 12–18-fold accelerations were record-
ed for (Æ)-1i, 1a, and 1h while excellent enantioselectivity
was maintained. On the other hand, only a slight accelera-
tion was recorded for 1g[32] and 1j,[35] and none was ob-
served for 1c[28] or 1 f.[31] The effect of IL1 on the enantiose-
lectivity was also dependent on the substrate: a remarkably
increased enantioselectivity was obtained for 1a, 1j, and 1l,
while only a small modification occurred for 1b.
Since it is anticipated that the supporting effect would be
general for lipases, we next investigated the IL1-supporting
effect of Candida rugosa lipase (CRL). Ueji and co-workers
recently reported that lyophilization of Candida rugosa
lipase in the presence of some ionic compounds resulted in
dramatic improvement of its enantioselectivity for the
esterification of 2-arylpropionic acid derivatives in an organ-
ic solvent system.[20c] Therefore, we chose the CRL-catalyzed
transesterification of 2-(4-ethylphenoxy)propionic acid ((Æ)-
4)[20] with n-butanol (nBuOH) as a model reaction[20] and
the results are shown in Table 5.
Substrate
lipase PS[a]
IL1-PS[a]
Specific
activity[c,d]
rate: 65
E 16
rate: 1000
E >200
15
98
1
rate: 100
E 39
rate: 9800
E 40
rate: 13
E >200
rate:14
E >200
rate: 1.0x10À2
E 199
rate: 11
E >200
1100
500
rate: 1.2x10À2
E >200
rate: 6
E >200
rate[b]: 52
E >200
rate[b]: 85
E >200
2
5
rate[b]: 32
E >200
rate[b]: 163
E >200
Both enantioselectivity and reaction rate were improved
when the reaction was carried out by using IL1-supported
CRL as catalyst (entries 3–5). The effect was significantly
dependent on the amount of the supporting IL1: high enan-
tioselectivity and marked acceleration were obtained for
IL1-CRL-b (entry 4) and IL1-CRL-c (entry 5), while only a
small activation was recorded for IL1-a. Reaction conver-
sion was 50% after 24 h reaction when IL1-CRL-b was
used as catalyst in iPr2O (entry 4), but it took 60 h at 26%
conversion when commercial CRL was used as catalyst
(entry 1). Interestingly, better enantioselectivity was ob-
tained when the reaction was carried out in the presence of
10 mol% (versus substrate) IL1, while the reaction rate de-
creased under the reaction conditions of entry 2. Further, it
was found that the IL1 support significantly stabilized CRL
in the organic solvent system. No drop in reactivity was ob-
served when IL1-CRL-c was placed in dry hexane for a
week at room temperature, while the reactivity was com-
pletely lost if commercial CRL was placed in hexane for the
same period. Since similar stabilization was observed for
PS-C, we expect that the stabilization effect of IL1 might be
general for various lipases.
rate[b]: 7.3
E >200
rate[b]: 133
E >200
18
rate: 2.6
E >200
rate: 31
E >200
12
26
6
rate[b]: 12
E 138
rate[b]: 300
E>200
rate: 3.2
E 12
rate: 19
E 123
rate: 1.1
E >200
rate: 12
E >200
11
[a] Rate: mhÀ1 mg(enzyme)À1
. Rate was determined by GC analysis.
N
[b] Rate was determined from the % conversion data, because the reac-
tion proceeded slowly. [c] Specific activity of IL1-PS versus PS-C.
[d] Specific activity was calculated by the rate of IL1-PS divided by that
of PS-C.
catalyzed transesterification occurred in [bmim]
solvent.[34] We attempted to demonstrate the recycling use of
the enzyme in the [bmim][C5F8] solvent system by using
A
Recycling use of IL1-PS: We recently reported the prepara-
tion of fluorine-substituted hydrophobic ionic liquid, 1-
butyl-3-methylimidazolium 2,2,3,3,4,4,5,5-octafluoropentyl sul-
AHCTREUNG
(Æ)-1j as substrate (Table 6). As shown in Table 6, both the
fate ([bmim]
A
reaction rate and enantioselectivity gradually decreased with
9232
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Chem. Eur. J. 2006, 12, 9228 – 9237

Ionic Liquids
FULL PAPER
Table 5. Results of esterification of 2-(4-ethylphenoxy)propionic acid
((Æ)-4) catalyzed by the ionic-liquid-supported CRL (Candida rugosa
lipase).
the recyclable use of lipase in [bmim]
PS as catalyst.
A
Investigation of the origin of this IL1-mediated activation:
To investigate the remarkable acceleration accomplished by
an ionic-liquid coating of the enzyme we next measured the
kinetic parameters of Burkholderia cepacia (PS-C)-catalyzed
transesterification of 1b. Enantiomerically pure alcohols
(R)-1b[16c,d] and (S)-1b[16c,d] were subjected to the lipase-cata-
lyzed transesterification by using vinyl acetate as acyl donor
in the iPr2O solvent system, and the progress of the reaction
was monitored by capillary gas chromatography to obtain
the initial rate (v0). The reaction was typically done as fol-
lows: dry iPr2O (2.0 mL) was added to alcohol (R)-1b or
(S)-1b together with the lipase (25 mg for PS-C or 7.0 mg
for IL1-PS). The resulting mixture was stirred at 358C and
sampled at one-minute intervals when IL1-PS was used as
catalyst. However, since the PS-C-catalyzed reaction pro-
ceeded slowly, we sampled at 30-minute intervals. The con-
centration of the acetate [S0] was systematically changed,
and the plot of v0 against [S0] afforded a typical saturation
curve.
Lipase[a]
Additive
t
Conversion
[%][b]
ee [%]
(R)-5[c]
E
[h]
value[b]
1
2
3
4
5
CRL
CRL
IL1-CRL-a
IL1-CRL-b
IL1-CRL-c
none
IL1[d]
none
none
none
60
96
32
24
24
26
14
27
50
36
62
98
92
>99
>99
5
96
32
>200
>200
[a] Lipase MY (Meito); IL1-CRL-a: IL1/MY=1.0 g:50 mg, IL1-CRL-b:
IL1/MY=1.0 g:500 mg, IL1-CRL-c: IL1/MY=1.0 g:1.0 g. [b] Calculated
by % ee of (R)-5 (eep) and % ee of (S)-4 (ees). E=ln
[(1Àc)(1Àeep)], here c means conversion which was calculated by the fol-
lowing formula: c=eep/(eep+ees). See ref. [23]. [c] Determined by HPLC
A
G
A
ACHTREUNG
ACHTREUNG
(Chiralcel OB, hexane/iPrOH=200:1). [d] 10 mol% of IL-1 was added.
Table 6. Results of recyclable use of PS-C and IL1-PS.
Table 7 shows the kinetic pa-
rameters for the PCL-cata-
lyzed transesterification of 3-
hydroxybutanenitrile (1b) by
using vinyl acetate as an acyl
donor. The ionic-liquid coating
significantly accelerated the re-
action by using two enantio-
mers of similar magnitude (ap-
Run
Lipase
t[h]
ee [%] of (R)-2j[a]
(yield [%])[b]
ee [%]of (S)-3j[a]
(yield [%])[b]
Conversion
[%][c]
E value[c]
proximately tenfold). Howev-
er, the difference in ratio of
Vmax values of (R)-1b/(S)-1b of
IL1-PS was slightly inferior to
that of PS-C (Table 4). It is
well recognized that the Km of
the lipase-catalyzed reaction
shows a generally large value,
indicating that the binding of
lipases to the substrates is
weak.[38] A large Km value was,
in fact, obtained by the present
G
ACHTREUNG
1
2
3
4
5
6
7
8
9
1st
PS-C
PS-C
PS-C
PS-C
24.0
24.0
24.0
24.0
24.0
2.5
4.5
6.0
8.0
13.0
98 (13)
96 (22)
93 (10)
93 (6)
25 (87)
16 (77)
10 (67)
9 (75)
20
14
10
9
148
62
31
31
16
165
65
72
87
82
2nd
3rd
4th
5th
1st
2nd
3rd
4th
5th
PS-C
88 (5)
5 (60)
5
IL1-PS
IL1-PS
IL1-PS
IL1-PS
IL1-PS
98 (32)
96 (22)
96 (30)
97 (20)
97 (18)
51 (62)
29 (66)
39 (61)
29 (77)
23 (78)
34
23
29
23
19
10
[a] Isolated yield. [b] The enantiomeric excess was determined by HPLC analysis by using OJ-H (1 4.6”
250 mm, hexane/iPrOH=9:1, 358C). [c] See ref. [23].
reaction.
A slightly reduced
Km was found in IL1-PS for
repetition of the reaction pro-
cess in the system when com-
Table 7. Kinetic parameters[a] for the PCL-catalyzed transesterification of 3-hydroxybutanenitrile (1b) by
using vinyl acetate as an acyl donor.
mercial PS-C was used as cata-
lyst (entries 1–5).[34] To our de-
light, the desired product was
obtained with excellent enan-
tioselectivity by using IL1-PS
as catalyst, and five repetitions
of this process showed no sig-
nificant drop in the reaction
rate (entries 6–10). We thus
succeeded in demonstrating
Substrate
Lipase
Vmax
Kcat
[minÀ1
Km
[m]
Kcat/Km
[mminÀ1 mg
(lipase)À1
]
G
]
[minÀ1 mÀ1
]
1
2
3
4
(R)-1b
(R)-1b
(S)-1b
(S)-1b
PS-C
IL1-PS
PS-C
3.1”10À1
3.0
2.0”104
2.0”105
4.6”102
6.0”103
1.6
2.5
1.3”104
8.0”104
7.5”102
2.1”104
7.0”10À3
9.1”10À2
6.0”10À1
2.9”10À1
IL1-PS
[a] Because of the heterogenous reaction, the nonlinear least-squares method was applied to the Michaelis–
Menten-type of equations: v0 =Vmax(E)mg[S0]/(Km+[S0]), in which Vmax is normalized by the weight of lipase
protein (E)mg
AHCTREUNG
.
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www.chemeurj.org
9233

T. Itoh et al.
the (S)-1b (entry 4) compared to that of PS-C (entry 3). As
a result, catalytic efficiency (Vmax/Km) between IL1-PS and
PS-C was modified: a slightly higher value was obtained for
IL1-PS than for PS-C. This corresponded to the increased
enantioselectivity of IL1-PS. As mentioned earlier, the cat-
ionic part of the coating materials significantly modified the
enantioselectivity of the enzyme (Table 2). We anticipated
that the cationic part of the ionic liquid might bind with the
lipase protein;this may cause conformational change of the
enzyme and contribute to increasing the difference of Km
between enantiomers. To confirm the binding of the ionic
liquid with the lipase protein, MALDI-TOF mass experi-
ments were carried out (Figure 1).
Figure 2. Scanning electron micrographs (SEM) of CF-PS, PS-C, Brij56-
PS, and seven types of ionic-liquid-coated PS, IL1–IL f at 1000-fold mag-
nitude.
Figure 1. Results of MALDI-TOF MS experiments on CF-PS and IL1-
PS.
The molecular peak of the lipase PS protein was found at
33295 Da, which coincided with the reported molecular
weight of Burkholderia cepacia lipase.[26] As clearly shown
in Figure 1, lipase PS protein made a complex with IL1 by
its appearance, such as, that at 34638 Da, though determina-
tion of the number of ionic-liquid molecules binding with
the enzyme was unsuccessful because IL1 has polymeric
parts of different molecular weight.[40]
Since IL1-PS is not soluble in iPr2O, it is assumed that the
interface property of lipase protein with the substrate mole-
cules might be important for the efficiency of the present
transesterification. Hence we looked at the surface proper-
ties of CF-PS, Brij56-PS, and ionic-liquid-coated lipase PS
(Figure 2). Significant differences were found in the surfaces
of CF-PS and IL1-PS. Lipase powders of ionic-liquid-coated
PS and Brij56-PS have a highly porous nature, while CF-PS
has a flat surface. IL1-PS appears to have a wider surface
area than that of native lipase PS (CF-PS) or commercial
PS-C, while all ionic-liquid-coated enzymes and Brij56-PS
appear to have similar-looking surface areas. Since signifi-
cant acceleration was obtained for these enzymes, the sur-
face-area expansion may take place during the lyophiliza-
tion process in the presence of ionic liquids or surfactant
Brij56;this may contribute to the production of highly
porous lipase powder. It is not surprising that the substrate
alcohol can access the enzyme more easily than the less-
porous CF-PS, and cause the rate acceleration of IL1-PS in
the transesterification. However, since enantioselectivity
was dependent on the nature of the coating material, partic-
ularly the cationic part of the ionic liquid, it is impossible to
explain the present lipase activation only by the surface-
area increase of the enzyme protein powder.
We currently assume that at least three factors are in-
volved in the origin of the modification of the present
9234
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Chem. Eur. J. 2006, 12, 9228 – 9237

Ionic Liquids
FULL PAPER
lipase-catalyzed reaction. One is the modified flexibility of
the enzyme by the ionic liquid. Ueji suggested that flexibili-
ty change of the lipase protein reflects on the stereoselectiv-
ity of the enzyme,[20] and an organic medium causes modifi-
cation of the flexibility of the lipase protein.[39] Since
MALDI-TOF mass experiments suggest that IL1 binds with
the enzyme protein, it seems possible that a polymer part of
the IL1 binding with the lipase protein may have a certain
impact on the flexibility of the lipase protein. The other
factor is the influence of the presence of water molecules
that might occur in IL1. The water content of the reaction
mixture (Table 2, entry 2), estimated by the Karl–Fischer
method, was 680 ppm. This level of water content does not
seem to be an important factor in increased enantioselectivi-
ty.[21b] From MALDI-TOF mass experiments on IL1, we es-
timated that this salt had at least 23 water molecules,[40]
though it was impossible to determine the exact number. A
small amount of water reportedly significantly influenced
the flexibility of enzymes and accelerated the transesterifica-
tion of lipase in the organic solvent system.[21] The third pos-
sible origin of the present lipase activation is that a cationic
part of the ionic liquid binds with lipase protein and causes
preferential modification of the enzyme conformation;this
may explain why the present activation of the enzyme de-
pends on the substrate.
Experimental Section
Reagents and solvents were purchased from common commercial sources
and were used as received or purified by distillation over appropriate
drying agents. 1H and 13C NMR spectra were recorded on a JEOL
JNM MH-500 or JNM MH-400 MHz spectrometer. Chemical shifts are
expressed in ppm downfield from tetramethylsilane (TMS) in CDCl3 as
an internal reference. IR spectra were obtained on SHIMADZU
FTIR 8000 spectrometers. Optical rotation was measured with a JASCO
DIP-370 digital polarimeter. The rate was determined by gas chromatog-
raphy analysis (Quadrex-bonded fused-silica methyl silicone,
1
0.25 mm”25 m, N2). The optical purity was determined by HPLC analy-
sis by using OD, OD-H, OB, AD, or OJ-H, and capillary gas chromatog-
raphy (Chiraldex G-TA, 1 0.25 mm”20 m, 1008C, He). MALDI-TOF
MS spectra were obtained by using a BRUKER AutoFLEX-T2 appara-
tus. SEM images were recorded by using a JEOL JSM-6390 LV instru-
ment.
1-Butyl-2,3-dimethylimidazolium chloride: A solution of 1,2-dimethylim-
G
N
red for 24 h under reflux conditions. After being cooled to RT, excess 1-
chlorobutane was removed under reduced pressure to give 1-butyl-2,3-di-
methylimidazolium chloride ([bdmim][Cl]) as a half-melted white solid
and it was used in the next reaction without further purification.
Ammonium poly[oxyethylene(n)] alkyl sulfate:
A mixture of Brij56
(poly[oxyethylene(10)] cetyl ether) (13.7 g, 20.0 mmol) and sulfamic acid
(1.94 g, 20.0 mmol) was stirred for 17 h at 1108C under argon and dried
under reduced pressure at 66.7 Pa, at 608C, for 3 h, to give ammonium
Brij56 sulfate as a white solid.
1-Butyl-2,3-dimethylimidazolium poly(oxyethylene) alkyl sulfate (IL1):
After the preparation of ammonium poly[oxyethylene(10)] cetyl sulfate
and 1-butyl-2,3-dimethylimidazolium chloride, these two crude products
were added to acetone (20 mL) and the mixture was stirred for 24 h at
RT. The ammonium chloride that precipitated was removed by filtration
through a sintered glass filter with a Celite pad. The filtrate was concen-
trated under vacuum for a little while, and then activated carbon was
added, and the mixture was stirred for 10 min. The activated carbon was
filtered through a sintered glass filter with a Celite pad and the filtrate
was filtered through an Al2O3 (neutral type I, activated) short column.
The filtrate was evaporated and dried under reduced pressure at 5 Torr,
for 24 h, at 608C, to give 1-butyl-2,3-dimethylimidazolium poly[oxyethyl-
Conclusion
We discovered that the novel ionic liquid [bdmim][cetyl-
PEG10-sulfate](IL1) worked as an excellent activator of li-
A
pase PS-catalyzed acetylation of various types of secondary
alcohols by using vinyl acetate as acyl donor in iPr2O, while
maintaining excellent enantioselectivity. More than 1000-
fold rate acceleration was accomplished for some substrates.
Lyophilization was essential to activate the lipase effectively
by the ionic liquid. Our activation system made it possible
to realize both increased enantioselectivity and rate acceler-
ation. In contrast, the previously established activation pro-
tocol, such as PEG coating of a lipase could accelerate the
reaction, but caused no significant increased enantioselectiv-
ity;also, using toluene as solvent sometimes provided in-
creased enantioselectivity for some substrates, but other
times caused a significant reduction in the reaction rate.
Since similar activation was observed for CRL, we expect
that the activation effect of IL1 might be general for various
lipases. We believe that this work represents not only a sig-
nificant advance in the manner of preparation of optically
active compounds by using an enzymatic reaction, but also
provides a new aspect in the application of an ionic liquid
for such a reaction. Since the present activation was signifi-
cantly dependent on the substrate, we are also hoping that
further optimization of the cationic part of the ionic liquids
may make it possible to apply the present protocol of lipase
activation for various broader types of substrates.
ene(10)] cetyl sulfate (13.6 g, 0.015 mol) as a yellowish solid at RT in
74% yield.
1-Butyl-2,3-dimethylimidazolium poly[oxyethylene(10)] cetyl sulfate
1
(IL1) (from Brij56): M.p. 35–378C; H NMR (500 MHz, CDCl3): d=0.80
(t, J=7.4 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H), 1.10–1.30 (m, 32H), 1.29–1.31
(m, 2H), 1.47–1.50 (m, 4H), 1.70–1.73 (m, 2H), 2.61 (s, 3H), 3.36 (t, J=
5.1 Hz, 4H), 3.49–3.62 (m, 34H), 3.81 (s, 3H), 4.00 (t, J=5.1 Hz, 2H),
4.07 (t, J=7.8 Hz, 2H), 7.24 (s, 1H), 7.38 ppm (s, 1H); 13C NMR
(125 MHz, CDCl3): d=9.48, 13.20, 13.77, 19.23, 22.30, 25.73, 28.98, 29.11,
29.23, 29.31, 31.35, 31.54, 35.06, 48.01, 61.20, 65.78, 69.68, 69.05, 70.19,
71.12, 72.26, 120.17, 122.65, 143.57 ppm;IR (neat): n˜ =2916, 2851, 1468,
1350, 1252, 1115, 951, 845 cmÀ1;MALDI-TOF MS (matrix: SA): m/z
calcd for C45H90N2O14S (average MW): 915.3;found: 1344.
Preparation of IL1-supported lipase PS (IL1-PS) by lyophilization:
Commercial lipase PS-C (1.0 g;Amano) was added to a potassium phos-
phate buffer (pH 7.2) solution (0.1m, 10 mL). The mixture was centri-
fuged at 3500 rpm for 5 min. IL1 (29.0 mg, approximately 3.1”
10À2 mmol) was dissolved into the resulting supernatant, that contains ap-
proximately 3.1”10À4 mmol of lipase PS protein, and the mixture was
lyophilized to give IL1-PS (344 mg) as a white powder. By using the
same procedure, thirteen types of supported lipase PS compounds were
prepared (for details, see Supporting Information).
Lipase PS-C-catalyzed acylation of (Æ)-1-phenylethanol (1a) with addi-
tive in iPr2O: Lipase PS-C (25 mg) was added to a solution of (Æ)-1a
(50 mg, 0.41 mmol), vinyl acetate (52.9 mg, 0.62 mmol), and an additive
(0.04 mmol) in iPr2O (2.0 mL) and the mixture was stirred at 358C. The
reaction course was monitored by capillary GC analysis and silica gel
Chem. Eur. J. 2006, 12, 9228 – 9237
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9235

T. Itoh et al.
TLC. Compounds (R)-2a and (S)-3a were obtained by preparative silica
gel thin-layer chromatography (TLC). The enantioselectivity was deter-
mined by HPLC analysis on a chiral column (Chiralcel OB, hexane/2-
propanol=8:1, 200:1).
Acknowledgements
The present work was supported by a Grant-in-Aid for Scientific Re-
search in the Priority Area “Science of Ionic Liquids” from the Ministry
of Education, Culture, Sports, Science and Technology, Japan and the
Asahi Glass Foundation.
1
(R)-2a: Rf =0.55 (hexane/ethyl acetate 4:1); H NMR (500 MHz, CDCl3):
d=1.47 (d, J=6.9 Hz, 3H), 2.00 (s, 3H), 5.81 (q, J=6.9 Hz, 1H), 7.19–
7.29 ppm (m, 5H); 13C NMR (125 MHz, CDCl3): d=21.25, 22.12, 72.22,
126.00, 127.77, 128.40, 141.59, 170.21 ppm;IR (neat): n˜ =2980, 1730,
1495, 1370, 1240, 1030, 940, 760 cmÀ1
.
[1] Reviews see: a) C.-H. Wong, G. M. Whitesides, Enzymes in Synthet-
ic Organic Chemistry, Pergamon, Oxford, 1994;b) U. T. Bornsche-
uer, R. J. Kazlauskas, Hydrolases in Organic Synthesis: Regio-and
Stereoselective Biotransformations, Wiley, Chichester 1999.
[2] Recently the name of this microorganism was changed from Pseudo-
monas cepacia to Burkholderia cepacia.
[3] Reviews see: a) M. Klivanov, Trends Biotechnol. 1997, 15, 97; b) T.
Itoh, Y. Takagi, H. Tsukube, Trends Org. Chem. 1997, 6, 1- 22;c) H.
Theil, Tetrahedron 2000, 56, 2905–2919.
[4] For example: a) S. Park, R. J. Kazlauskas, J. Org. Chem. 2001, 66,
8395–8401;b) T. Maruyama, S. Nagasawa, M. Goto, Biotechnol.
Lett. 2002, 24, 1341–1345;c) U. Bora, C. J. Saikia, A. Chetia, A. K.
Mishra, B. S. D. Kumar, R. C. Boruah, Tetrahedron Lett. 2003, 44,
9099–9102.
[5] a) Y. Okahata, K. Ijiro, J. Chem. Soc. Chem. Commun. 1988, 1392–
1394;b) Y. Okahata, Y. Fujimoto, K. Ijiro, Tetrahedron Lett. 1988,
29, 5133–5134;c) Y. Okahata, K. Ijiro, Bull. Chem. Soc. Jpn. 1992,
65, 2411–2420;d) W. Tsuzuki, Y. Okahata, O. Katayama, T. Suzuki,
J. Chem. Soc. Perkin Trans. 1 1991, 1245–1247;e) Y. Okahata, Y.
Fujimoto, K. J. Ijiro, Org. Chem. 1995, 60, 2244–2250;f) Y. Okaha-
ta, A. Hatano, K. Ijiro, Tetrahedron: Asymmetry 1995, 6, 1311–
1322;g) Y. Okahata, T. Mori, Trends Biotechnol. 1997, 15, 50–54;
h) “Enzymes in Non-aqueous Solvents: Methods and Protocols” T.
Mori, Y. Okahata, Methods Biotechnol. 2001, 15, 83–94;i) T. Mori,
S. Kishimoto, K. Ijiro, A. Kobayashi, Y. Okahata, Biotechnol.
Bioeng. 2001, 76, 157–163;j) H. Nishino, T. Mori, Y. Okahata,
Chem. Commun. 2002, 2684–2685.
[6] a) M. T. Reetz, A. Zonta, J. Simpelkamp, Angew. Chem. 1995, 107,
373–376; Angew. Chem. Int. Ed. Engl. 1995, 34, 301–303;b) M. T.
Reetz, A. Zonta, J. Simpelkamp, W. Kçnen, Chem. Commun. 1996,
1397–1398;c) M. T. Reetz, W. Wiesenhçfer, G. Franciꢁ, W. Leitner,
Adv. Synth. Catal. 2003, 345, 1221–1228.
[7] a) I. Mingarro, C. Abad, L. Braco, Proc. Natl. Acad. Sci. USA 1995,
92, 3308–3312;b) I. Mingarro, H. Gonzalez-Navarro, L. Braco, Bio-
chemistry 1996, 35, 9935–9944;c) H. Gonzalez-Navarro, L. Braco, J.
Mol. Catal. B 1997, 3, 111–119;d) H. Gonzalez-Navarro, L. Braco,
Biotechnol. Bioeng. 1998, 59, 122–127.
(S)-3a: Rf =0.25 (hexane/ethyl acetate=4:1); 1H NMR (500 MHz,
CDCl3): d 1.43 (d, J=6.4 Hz, 3H), 1.75 (s, 1H;OH), 4.83 (q, J=6.4 Hz,
1H), 7.28–7.30 ppm (m, 5H); 13C NMR (125 MHz, CDCl3): d=25.01,
70.13, 125.30, 127.26, 128.33, 145.76 ppm;IR (neat): n˜ =3330, 3030, 2970,
2890, 1490, 1450, 1010, 900 cmÀ1
.
IL1-PS-catalyzed acylation of 1-phenylethanol (1a) in iPr2O: IL1-PS
(7.0 mg) was added to a solution of (Æ)-1a (50 mg, 0.41 mmol) and vinyl
acetate (52.9 mg, 0.62 mmol) in iPr2O (2.0 mL) and the mixture was stir-
red at 358C. The reaction course was monitored by capillary GC analysis
and silica gel TLC. Compounds (R)-2a and (S)-3a were obtained by
preparative silica gel thin-layer chromatography (TLC). The enantiose-
lectivity was determined by HPLC analysis by using a chiral column
(Chiralcel OB, hexane/2-propanol=8:1, 200:1).
1-Butyl-3-methylimidazolium
2,2,3,3,4,4,5,5-octafluoropentyl
sulfate
([bmim] mixture of 2,2,3,3,4,4,5,5-octafluoropentanol
A
A
(33.0 g, 0.14 mol) and sulfamic acid (13.6 g, 0.14 mol) was stirred at
1308C for 24 h under argon atmosphere and was cooled to RT to give
ammonium 2,2,3,3,4,4,5,5-octafluoropentyl sulfate as a white precipitate.
The ammonium salt was washed with hexane three times and evaporated
to dryness. [bmim][Cl] (23.6 g, 0.135 mol) was added to a solution of the
salt in acetone (135 mL) and the resulting solution was stirred 24 h at RT
to form ammonium chloride (NH4Cl) as a white precipitate. Precipitated
NH4Cl was removed by filtration through a sintered glass filter with a
Celite pad and the filtrate was concentrated under vacuum to give
[bmim][C5F8] as viscous oil. This was washed with a mixed solvent of
E
hexane and ethyl acetate (4:1) and water, then diluted with acetone, and
treated with activated charcoal. The activated charcoal was removed by
filtration through a sintered glass filter with a Celite pad and the filtrate
was filtered through an Al2O3 (neutral type I, activated) short column,
then was finally lyophilized to give [bmim][C5F8] (40.2 g, 0.089 mol) as
T
yellowish oil in 66% yield: 1H NMR (500 MHz, CDCl3): d=0.95 (t, J=
7.3 Hz, 3H), 1.32–1.40 (m, 2H), 1.83–1.89 (m, 2H), 3.98 (s, 3H), 4.21 (t,
J=7.4 Hz, 2H), 4.45 (t, J=14.7 Hz, 2H), 5.99–6.23 (m, 1H), 7.32 (s, 1H),
7.39 (s, 1H), 9.18 ppm (s, 1H); 13C NMR (125 MHz, CDCl3): d=12.99,
19.16, 31.78, 36.02, 49.65, 62.95 (t,
30.5 Hz, JC,F =253.3 Hz;2-CF 2), 109.93 (2t, JC,F =25.8 Hz, JC,F =261.3 Hz;
3-CF2), 110.63 (2t, JC,F =30.5 Hz, JC,F =263.2 Hz;4-CF ), 114.85 (2t, JC,F
30.5 Hz, JC,F =253.3 Hz;CF 2H), 122.07, 123.62, 136.56 ppm; 19F NMR
(470 MHz, CDCl3): d=24.31 (t, J=51.8 Hz, 2F), 31.61 (s, 2F), 36.25 (s,
2F), 41.57 ppm (t, J=11.5 Hz, 2F);IR (neat): n˜ =2880, 1510, 1460, 1270,
1240, 1170, 1130, 1040 cmÀ1;elemental analysis calcd (%) for
C13H18F8N2O3S: C 35.95, H 4.18, N 6.45;found: C 35.88, H 4.19, N 6.46.
JC,F =24.9 Hz), 107.53 (2t, JC,F =
[8] a) Y. L. Khmelnitsky, S. H. Welch, D. S. Clark, J. S. Dordick, J. Am.
Chem. Soc. 1994, 116, 2647–2648;b) M. T. Ru, S. Y. Hirokane, A. S.
Lo, J. S. Dordick, J. A. Reimer, S. S. Clark, J. Am. Chem. Soc. 2000,
122, 1565–1571;c) J. P. Lindsay, D. S. Clark, J. S. Dordick, Biotech-
nol. Bioeng. 2004, 85, 553–560.
[9] J. K. Lee, M.-J. Kim, J. Org. Chem. 2002, 67, 6845–6847.
[10] For recent reviews for reactions in ionic-liquid solvent systems see:
a) Ionic Liquids in Synthesis (Eds.: P. Wasserscheid, T. Welton),
Wiley-VCH, 2003;b) “Ionic Liquids as Green Solvents”: ACS Symp.
Ser. 2003, 856, whole volume;c) N. Jain, A. Kumar, S. Chauhan,
S. M. S. Chauhan, Tetrahedron 2005, 61, 1015–1060.
[11] For our examples of lipase-catalyzed reaction in the ionic-liquid sol-
vent system see: a) T. Itoh, E. Akasaki, K. Kudo, S. Shirakami,
Chem. Lett. 2001, 262–263;b) T. Itoh, E. Akasaki, Y. Nishimura,
Chem. Lett. 2002, 154–155;c) T. Itoh, Y. Nishimura, M. Kashiwagi,
M. Onaka ACS Symp. Ser. 2003, 856, 251–261;d) T. Itoh, N. Ouchi,
S. Hayase, Y. Nishimura, Chem. Lett. 2003, 32, 654–655;e) T. Itoh,
Y. Nishimura, N. Ouchi, S. Hayase, J. Mol. Catal. B 2003, 26, 41–45;
f) T. Itoh, N. Ouchi, Y. Nishimura, S.-H. Han, N. Katada, M. Niwa,
M. Onaka, Green Chem. 2003, 5, 494–496.
=
2
Lipase PS-C-catalyzed acylation of 4-phenyl-3-butene-2-ol (1j) in
[bmim]
added to
0.35 mmol) and vinyl acetate (50.2 mg, 0.58 mmol) in [bmim]
ACHTREUNG
a
AHCTREUNG
(1.0 mL) and the mixture was stirred at 358C for 24 h. The reaction
course was monitored by capillary GC analysis and silica gel TLC. Dieth-
yl ether (1.5 mL) was added to the reaction mixture of to form biphasic
layers and the product acetate (R)-2j and alcohol (S)-3j were isolated
from the ether layer. It was essential to repeat the extraction with ether
from the reaction mixture ten times. Since the lipase remained in the
ionic liquid layer, it was possible to use the lipase repeatedly;the ionic
layer was placed under reduced pressure at RT for 5 h to remove the
ether and (Æ)-1j (59.0 mg, 0.35 mmol) and vinyl acetate (50.2 mg,
0.58 mmol) was added to the resulting ionic layer and the mixture was
stirred at 358C.
[12] Preliminary results of this project, see: T. Itoh, S. Han, Y. Matsushi-
ta, S. Hayase, Green Chem. 2004, 6, 437–439.
[13] Early examples for lipase-catalyzed reactions in a pure ionic liquid
solvent system, see: a) R. M. Lau, F. van Rantwijk, K. R. Seddon,
9236
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9228 – 9237

Ionic Liquids
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Received: June 26, 2006
Published online: October 9, 2006
Chem. Eur. J. 2006, 12, 9228 – 9237
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9237