Biocatalysis in Ionic Liquids: Markedly Enhanced Enantioselectivity of Lipase

Article abstract of DOI:10.1021/ol015824f

matrix presented Lipase-catalyzed transesterifications in ionic liquids proceeded with markedly enhanced enantioselectivity. It was observed that lipases were up to 25 times more enantioselective in ionic liquids than in conventional organic solvents.

Full text of DOI:10.1021/ol015824f

ORGANIC
LETTERS
2
001
Vol. 3, No. 10
507-1509
Biocatalysis in Ionic Liquids: Markedly
Enhanced Enantioselectivity of Lipase
1
Kwang-Wook Kim, Boyoung Song, Min-Young Choi, and Mahn-Joo Kim*
National Research Laboratory of Chirotechnology, Department of Chemistry, DiVision
of Molecular and Life Sciences, Pohang UniVersity of Science and Technology,
San 31 Hyojadong, Pohang, Kyungbuk 790-784, Korea
mjkim@postech.ac.kr
Received March 12, 2001
ABSTRACT
Lipase-catalyzed transesterifications in ionic liquids proceeded with markedly enhanced enantioselectivity. It was observed that lipases were
up to 25 times more enantioselective in ionic liquids than in conventional organic solvents.
5
Room-temperature ionic liquids are attracting growing inter-
est as alternative reaction media for chemical transforma-
tions.1 One of several advantages of ionic liquids is that
they are environmentally benign since they have no detect-
able vapor pressure. Accordingly, they are emerging as novel
replacements for volatile organic solvents in industrial
organic synthesis. They are particularly promising as solvents
for catalysis. Their use can enhance activity, selectivity, and
stability of catalysts. Most of the studies in this area have
so far focused on transformations using transition-metal
in transesterifications. It was observed that lipases were up
to 25 times more enantioselective in ionic liquids than in
conventional organic solvents.
,2
+
Two ionic liquids, [EMIM]-[BF
4
] (1, [EMIM] ) 1-ethyl-
6
+
3
-methylimidazolium) and [BMIM]-[PF
6
] (2, [BMIM] )
3
catalysts. Very recently, two groups have reported the use
4
of ionic liquids as solvents in enzymatic reactions.
(
3) (a) Chauvin, Y.; Mussmann, L.; Olivier, H. Angew. Chem., Int. Ed.
Engl. 1995, 34, 2698. (b) Dullis, J. E L.; Suarez, P. A. Z.; Einloft, S.; de
Souza, R. F.; Dupont, J.; Fisher, J.; DeCian, A. Organometallics 1998, 17,
Erbeldinger et al. used ionic liquid in the synthesis of (Z)-
4a
aspartame using thermolysin. Lau et al. reported that lipase-
catalyzed alcoholysis, ammonolysis, and perhydrolysis in
ionic liquids and reaction rates were generally comparable
8
15. (c) Carmichael, A. J.; Earle, M. J.; Holbrey, J. D.; McCormac, P. B.;
Seddon, K. R. Org. Lett. 1999, 1, 997. (d) Dyson, P. J.; Ellis, D. J.; Parker,
D. G.; Welton, T. Chem. Commun. 1999, 25. (e) Chen, W.; Xu, L.;
Chatterton, C.; Xiao, J. Chem. Commun. 1999, 1247. (f) Xu, L,; Chen. W.;
Xiao, J. Organometallics 2000, 19, 1123. (g) Brasse, C. C.; Englert, U.;
Salzer, A. Organometallics 2000, 19, 3818. (h) Song, C. E.; Roh, E. J.
Chem. Commun. 2000, 837. (i) Owens, G. S.; Abu-Omar, M. M. Chem.
Commun. 2000, 1165. (j) Carmichael, A. J.; Haddleton, D. M.; Bon, S. A.
F.; Seddon, K. R. Chem. Commun. 2000, 1237. (k) Mathews, C. J.; Smith,
P. J.; Welton, T. Chem. Commun. 2000, 1249. (l) Song, C. E.; Shim, W.
H.; Roh, E. J.; Choi, J. H. Chem. Commun. 2000, 1695. (m) Song, C. E.;
Oh, C. R.; Roh, E. J.; Choo, D. J. Chem. Commun. 2000, 1743.
4
b
with, or better than, those observed in organic media. As
a part of our “green chemistry” research program, we became
interested in ionic liquids as alternative solvents for biotrans-
formations using cell-free enzymes. We herein wish to report
the preliminary results from our studies that the use of ionic
liquids enhanced significantly the enantioselectivity of lipase
(
4) (a) Erbeldinger, M.; Mesiano, A. J.; Russel, A. Biotechnol. Prog.
(
1) Reviews: (a) Seddon, K. R. J. Chem. Technol. Biotechnol. 1977,
8, 351. (b) Welton, T. Chem. ReV. 1999, 99, 2071. (c) Wasserscheid, P.;
Wilhelm, K. Angew. Chem., Int. Ed. 2000, 39, 3772.
2) (a) Ford, W. T.; Hauri, R. J.; Hart, D. J. J. Org. Chem. 1973, 38,
916. (b) Bolkan, S. A.; Yoke, J. T. Inorg. Chem. 1986, 25, 3587. (c) Wilkes,
2000, 16, 1131. (b) Lau, R. M.; Van Rantwijk, F.; Seddon, K. R.; Sheldon,
R. A. Org. Lett. 2000, 2, 4189.
6
(5) During the preparation of this manuscript, a report describing the
improved enantioselectivity of lipase in ionic liquids has appeared (Schoefer,
S. H.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001, 425).
(6) For the preparation of 1, see: (a) Wilkes, J. S.; Levisky, J. A.; Wilson,
R. A.; Hussey, C. L. Inorg. Chem. 1982, 21, 1263. (b) Fuller, J.; Carlin, R.
T.; De long, H. C.; Haworth, D. J. Chem. Soc., Chem. Commun. 1994,
299.
(
3
J. S.; Zaworotko, M. J. J. Chem. Soc., Chem. Commun. 1992, 965. (d) Fuller,
J.; Carlin, R. T.; Osteryoung, R. A. J. Electrochem. Soc. 1997, 144, 3881.
e) Larsen, A. S.; Holbrey, J. D.; Tham, F. S.; Reed, C. A. J. Am. Chem.
(
Soc. 2000, 122, 7264.
1
0.1021/ol015824f CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/04/2001

7
1
-butyl-3-methylimidazolium), were tested as media for
lipase-catalyzed transesterification (Scheme 1) and compared
Table 1. The Enantioselectivities for the Lipase-Catalyzed
Transesterification in Organic Solvents and Ionic Liquids
a
entry substrate lipase medium
ees
eep
E
Scheme 1. Lipase-Catalyzed Transesterification
1
2
3
4
5
6
7
8
9
0
1
2
3
4
3a
3b
3c
3d
CALB THF
toluene
0.916
0.976
0.915
0.955
0.958
0.990
141
207
648
>967
26
187
651
155
56
158
183
>450
150
85
1
2
0.789 >0.995
CALB THF
0.803
0.975
0.941
0.677
0.182
0.420
0.397
0.827
0.954
0.989
0.941
0.958
0.981
0.984
toluene
1
2
PCL
PCL
THF
toluene
1
2
1
1
1
1
1
0.123 >0.995
with two organic solvents, THF and toluene, in terms of
enzyme enantioselectivity. The evaluations of enzyme enan-
tioselectivity in these media were carried out with alcohols
THF
0.420
0.418
0.499
0.980
0.965
0.981
toluene
1
2
15
16
172
3
a-d as the substrates in the presence of vinyl acetate at
0.854 >0.995 >1000
room temperature. Two lipases, Candida antarctica lipase
a
On the basis of analyses by HPLC using a chiral column. Analytical
conditions: Chiralcel OD, hexane/2-propanol ) 95/5 (3a), 98/2 (3b), 90/
10 (3d, 4d), flow rate ) 1.0 (3a,b), 0.5 mL/min (3d, 4d), UV 217 (3a,b),
8
B (CALB, immobilized) and Pseudomonas cepacia lipase
9
(
PCL, native), were chosen as the enzymes. The enantio-
2
50 nm (3d, 4d); Whelk-O1, hexane/2-propanol ) 99/1 (3c, 4a-c), flow
rate ) 0.5 (4a,b), 1.0 mL/min (3c, 4c, UV 217 (3c, 4a-c).
selectivity of CALB was evaluated for the reactions of 3a
and 3b, and the enantioselectivity of PCL was evaluated for
the reactions of 3c and 3d.
In typical experiments, an enzymatic reaction was per-
formed with a solution containing substrate (0.15 mmol),
lipase (20 mg), and vinyl acetate (1.5-3 equiv) in solvent
of 3b showed a different pattern in enantioselectivity (entries
5-8). The highest enantioselectivity (E ) 651) was observed
in 1, but the enantioselectivity in 2 was moderate (E ) 155)
and comparable to that in toluene. The lowest enantioselec-
tivity was observed in THF. Accordingly, the enantioselec-
tivity enhancements were 25-fold between THF and 1, 3.5-
fold between toluene and 1, and 6-fold between THF and 2.
The PCL-catalyzed transesterifications of 3c and 3d
proceeded with high enantioselectivity (E ) >450 and
(1 mL) at room temperature. After the reaction reached 10-
50% completion, the enzymes were removed by filtration
and the resulting solution was concentrated. In the case of
ionic liquid solution, the solution mixture was first extracted
with ethyl ether and the ethereal phase was concentrated.
The organic residues were subjected to silica gel chroma-
tography to obtain unreacted substrate and acetylated product.
Their optical purities were then determined by HPLC using
a chiral column, which allowed us to measure the enantio-
meric excess (ee) up to >99.5%. The E values were
>1000) in ionic liquid 2 (entries 12 and 16). The enantio-
selectivities in ionic liquid 1 were modest (E ) 183 and 172)
but comparable to the best (E ) 158 and 150) in organic
solvents (entries 10, 11, 13, and 15). Accordingly, the use
of 2 enhanced the enantioselectivity by 3-8 times for the
reaction of 3c and by 7-12 times for the case of 3d.
The enantioselectivities of lipases, in general, were higher
in hydrophobic 2 than in hydrophilic 1 except for the reaction
of 3b. In several cases, the lipase enantioselectivity in ionic
liquid reached a synthetically desirable level (E ) >400,
entries 4, 7, 12, and 16). As an illustrative example, the
reaction of 3d corresponding to the entry 16 was performed
calculated using the equation, E ) ln[1 - c(1 + ee
p
)]/ln[1
10
-
c(1 - ee
p
)], where c ) ee
s
/(ee
s
+ ee
p
). The results are
given in Table 1.
The transesterification of 3a catalyzed by CALB proceeded
with better enantioselectivity in ionic liquids than in organic
solvents (Table 1, entries 1-4). The best enantioselectivity
(E ) >967) was observed for the reaction in 2, which was
5-7 times higher than for those in organic solvents. The
high enantioselectivity (E ) 648) was observed for the
reaction in 1, which was 3-4 times higher compared to those
in organic solvents. The CALB-catalyzed transesterification
11
twice on a 0.6 mmol scale. In the first run, the reaction
(11) Experimental detail: 3d (109 mg, 0.6 mmol), vinyl acetate (1.8
mmol), and PCL (218 mg) were mixed with ionic liquid 2 (3 mL), and the
resulting heterogeneous mixture was stirred at 25 °C. When the reaction
reached slightly over 50% completion (48 h), the reaction mixture was
extracted with ethyl ether (5 times with a 20 mL portion). The ethereal
phase was concentrated and subjected to silica gel chromatography to
provide the unreacted substrates ((R)-3d, 47.3 mg, 0.26 mmol, 43%, >99.5%
ee) and the acetylated products ((S)-4d, 63 mg, 0.28 mmol, 47%, 97.7%
ee). The second reaction was performed under the same conditions, except
that the reaction was carried to about 46% completion (36 h), to afford the
unreacted substrates (46.5 mg, 0.256 mmol, 43%, 85.8% ee) and the
acetylated product (56.9 mg, 0.255 mmol, 42%, >99.5% ee). The optical
purities were determined by HPLC using a chiral column. See the footnote
in Table 1 for the HPLC conditions.
(
7) For the preparation of 2, see: (a) Suarez, P. A. Z.; Dullius, J. E. L.;
Einloft, S.; De Souza, R. F.; Dupont, J. Polyhedron 1996, 15, 1271. (b)
Bonh oˆ te, P.; Dias, A.-P.; Papageorgiou, N.; Kalyanasundaram, K.; Gr a¨ tzel,
M. Inorg. Chem. 1996, 35, 1168.
(
8) We used the enzyme from Roche, whose trade name is Chirazyme
L-2, c-f, C3, lyo. Its price is 406 DM for 150 kU.
9) This enzyme is available from some commercial suppliers such as
(
Fluka, Roche, and Amano. We used the one provided by Amano, whose
price is not available. The price of the Roche enzyme (trade name,
Chirazyme L-1, Lyo) is 406 DM for 5.0 MU.
(10) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294.
1508
Org. Lett., Vol. 3, No. 10, 2001

was carried to slightly over 50% completion to obtain the
unreacted substrates of >99.5% ee in 43% yield. In the
second run, the reaction was carried to 46% completion to
get the acetylated products of >99.5% ee in 42% yield.
Accordingly, we should wait until further systematic studies
disclose the effects of these factors with some detail in the
near future.
In summary, this work has demonstrated that ionic liquids
can serve as the solvents for lipase-catalyzed transesterifi-
cations with an advantage of enhancing the enantioselectivity,
suggesting that they have great potential as alternative media
for biocatalysis and biotransformations. Several ionic liquids
including those used in this work are commercially avail-
It was observed that in most cases the activities of lipases
in ionic liquids were as good as or slightly lower than in
12
organic solvents. One exceptional case was the reaction of
c in 2, which proceeded at much lower rate. An advantage
3
of ionic liquids as solvents is that they can be readily reused
together with catalysts. For the CALB-catalyzed reaction of
1
3
able. The lipase reactions in ionic liquids can be scaled up
14
without a major difficulty. We believe that the use of ionic
liquids now opens up a new field in nonaqueous enzymology
as the use of organic solvents did in the early 1980s.15
3a in 2, we were able to reuse ionic liquid and enzyme twice
without loss in enantioselectivity and reactivity after the first
run.
All of these results clearly prove that the lipase-catalyzed
transesterifications took place effectively in ionic liquids 1
and 2, in some cases with impressively high enantioselec-
tivity. Now the question is why lipases show such high
enantioselectivity in ionic liquids. At this time, no satisfactory
answers are available since the enzyme enantioselectivity
could be affected by several factors, including the nature of
medium, the diffusion rates of substrate and product, the
water content, and the conformational rigidity of enzymes.
Acknowledgment. This work was supported by the
Korean Ministry of Science and Technology. We thank the
Korean Ministry of Education for a postdoctoral fellowship
to K.W.K. and a graduate fellowship to B.S.
OL015824F
(13) From Solvent Innovation GMBH, Cologne. The prices of 1 and 2
are, respectively, 529.30 and 647.50 DM for 100 mL. In this work, we
used chemically prepared ionic liquids. See ref 7.
(14) When the reaction corresponding to entry 4 in Table 1 was scaled
up by 10-fold (3a, 225 mg, 1.5 mmol), the same level of enantioselectivity
was realized.
(
12) The activities of lipases decreased roughly in the following orders:
(15) Reviews: (a) Klivanov, A. M. Chemtech 1986, 16, 354. (b)
Klibanov, A. M. Acc. Chem. Res. 1989, 23, 114. (c) Enzymatic Reactions
in Organic Media; Koskinen, A. M. P., Klibanov, A. M., Eds.; Blackie
Academic & Professional: Glasgow, 1996.
toluene > THF≈ 1 ≈ 2 for the reaction of 3a, toluene > THF ≈ 1 > 2 for
the reaction of 3b, toluene > THF ≈ 1 >> 2 for the reaction of 3c, and
toluene ≈ THF ≈ 2 > 1 for the reaction of 3d.
Org. Lett., Vol. 3, No. 10, 2001
1509