Enzyme catalysis in ionic liquids: Lipase catalysed kinetic resolution of 1-phenylethanol with improved enantioselectivity

Article abstract of DOI:10.1039/b009389k

Lipases show good activity and, in some cases, improved enantioselectivity when employed in pure ionic liquids for dynamic kinetic resolution of 1-phenylethanol by trans-esterification.

Full text of DOI:10.1039/b009389k

Enzyme catalysis in ionic liquids: lipase catalysed kinetic resolution of
1-phenylethanol with improved enantioselectivity
Sonja H. Schöfer,a Nicole Kaftzik,a Peter Wasserscheidb and Udo Kragl*a
a Rostock University, Dept. of Chemistry, 18051 Rostock, Germany. E-mail: udo.kragl@chemie.uni-rostock.de
b RWTH Aachen, Institut für Technische Chemie und Makromolekulare Chemie, 52066 Aachen, Germany
Received (in Liverpool, UK) 16th November 2000, Accepted 26th January 2001
First published as an Advance Article on the web 13th February 2001
Lipases show good activity and, in some cases, improved
enantioselectivity when employed in pure ionic liquids for
dynamic kinetic resolution of 1-phenylethanol by trans-
esterification.
miscible co-solvent suppresses the secondary hydrolysis of the
formed product resulting in doubling the yield to almost 60%!
The reaction investigated here yields two products: the
remaining alcohol (S)-6 or the formed acetate (R)-8. Unless
stated otherwise the enantioselectivity is always given for the
(R)-acetate. When the enzyme shows low enantioselectivity it is
not possible to reach high ee-values for the (R)-acetate whereas
for the (S)-alcohol high ee is possible at the expense of the
yield.
A set of nine lipases and two esterases (Roche Diagnostics
Chirazyme Screening Set 2) was screened for activity in ten
different ionic liquids.17 The results were compared with the
reaction performed in methyl tert-butyl ether (MTBE) as
solvent. MTBE is widely used as solvent for transesterification
in industry and academia. Therefore it was used for this study
despite the fact that some other solvents might be favourable as
well.
The data are summarised in Table 1. Under the conditions
employed the two pig liver esterases showed no activity. Best
results were obtained with Candida antarctica lipase B (L-2)
and Pseudomonas sp. lipase (L-6) in several ionic liquids. There
is no ‘best ionic liquid’ in general, but [BMIM](CF3SO2)2N
seems to have some advantages. Surprisingly, with the lipases
from Pseudomonas sp. and Alcaligenes sp. (L-10) the enantio-
selectivity for the formation of the acetate (R)-8 is improved to
a large extent compared to the reaction in MTBE. For the
Candida antarctica lipase A (L-5) the opposite is observed. The
increase of enantioselectivity is reproducible under different
conditions. It is likely that the ILs will interact with charged
residues found in or near the active centre of the enzyme. For
two of the enzymes, L-2 and L-6 concentrations and enantiose-
lectivity were followed as a function of time in MTBE,
[BMIM]CF3SO3 and [BMIM](CF3SO2)2N. The reaction ve-
locity is equal in both media, MTBE or IL.
One of the major advantages of ILs is that they are not
volatile. Therefore it is possible to remove the products by
distillation and repeat the catalytic cycle after addition of fresh
substrate. This was investigated for L-2 in [BMIM]-
(CF3SO2)2N. The lipase shows good thermal stability up to 100
°C in MTBE as well as in the IL. Unfortunately, the substrate
chosen and the product have boiling points around 200 °C at
atmospheric pressure. Therefore, even at a pressure of 0.06
mbar a temperature of 85 °C was necessary to remove the
reactants. But the enzyme suspended in the IL could be reused
three times with less than 10% loss of activity per cycle. The
enantioselectivity was not influenced. Certainly the recycling of
the ionic liquid–enzyme system would be easier for other
substrates as well as on a larger scale. These aspects as well as
the improved enantioselectivity are subject to further studies.
Additionally, other factors such as the water content, the
viscosity or the question of which of the ions is responsible for
the effects will be investigated as well.
Today, more than 100 one-step biotransformations making use
of whole cells or isolated enzymes are employed on an
industrial scale, including a very recent process established by
BASF for the kinetic resolution of chiral amines using
lipases.1,2 On a lab scale more than 13000 enzyme-catalysed
reactions have been described.3,4 Nevertheless, there are still
problems with substrate solubility, yield or (enantio-)selectiv-
ity. Some progress has been made by addition of organic
solvents,5 addition of high salt concentrations6 or use of
microemulsions7 or supercritical fluids.8 Recently, ionic liquids
(IL) have gained increasing attention for performing all types of
reactions with sometimes remarkable results.9–11 By modifica-
tion of the cation and anion their properties can be tuned in
many ways. For all catalytic processes, there are basically three
modes of operation: use of the IL as a co-solvent, as pure solvent
or in a biphasic system. After the first trials using ethylammon-
ium nitrate in salt water mixtures more than 15 years ago,12
recently the first results of the use of pure ILs as a reaction
medium for enzymatic reactions have been published.13,14
A
biphasic system containing an IL for in situ product extraction
for a whole cell process has been described as well.15
In this paper we report our results about the application of
lipases for an enantioselective reaction in pure ILs based on
1-butyl-3-methylimidazolium (BMIM) ions such as
[BMIM]PF6 1, [BMIM]CF3SO3 2 and [BMIM](CF3SO2)2N 3
or on N-butylpyridinium ions such as [4-MBP]BF4 4. As a
model system the kinetic resolution of rac-1-phenylethanol 6 by
transesterification with vinyl acetate 7 was investigated
(Scheme 1). Our results on the b-galactosidase catalysed
synthesis of N-acetyllactosamine are reported elsewhere.16 In
that case, addition of 25% v/v of [MMIM]MeSO4 5 as a water-
The results presented here clearly demonstrate the potential
of ionic liquids for enzymatic biotransformations. The varia-
tions possible for tailor-made solvents may have a similar
impact as the pioneering work of the use of enzymes in pure
organic solvents.18
Scheme 1
DOI: 10.1039/b009389k
Chem. Commun., 2001, 425–426
This journal is © The Royal Society of Chemistry 2001
425

View Online
Table 1 Results of the screening of lipases in various ionic liquids. Conversion (%) of rac-6 and ee’s (%) of (R)-8 (in brackets) are given. Conversion and
ee were estimated by HPLC. Purified L-3 is also part of the Roche Chirazyme screening set
Enzymes
Solvent
L-2h
L-3i
L-3 purified L-5j
L-6k
L-7l
L-8m
L-9n
L-10o
MTBE
[BMIM]PF6
[NMIM]PF6
[BMIM]BF4
[HMIM]BF4
[OMIM]BF4
[4-MBP]BF4
[BMIM]CF3SO3
[BMIM](CF3SO2)2Na
43 ( > 98)
< 5
10 ( > 98)
< 5
13 (47)
< 5
7 (70)
41 ( > 98) < 5
0
< 5
0
0
11 (22)
53 (84)
0
45 ( > 98)
< 5
17 ( > 98) < 5
10 ( > 98)
< 5
11 ( > 98)
< 5
0
29 ( > 98) > 98 (0)
a
b
10 (37)
41 (71)
< 5
27 (34)
59 (13)
> 98 (3)
44 (45)
> 98 (0)
< 5
44 (77)
33 ( > 98)
68 (14)
a
c
7 (53)
0
< 5
9 ( > 98) < 5
50 ( > 98) < 5
47 ( > 98)
0
0
< 5
< 5
0
< 5
< 5
60 (81)
10 ( > 98)
0
< 5
< 5
< 5
< 5
26 ( > 98)
50 ( > 98)
15 ( > 98)
70 (82)
d
e
41 ( > 98) < 5
46 ( > 98) < 5
50 ( > 98) < 5
< 5
< 5
a
9 ( > 98) < 5
12 ( > 98) 40 ( > 98)
50 ( > 98)
10 (69)
8 ( > 98)
89 (15)
f
[MMIM]MeSO4
[EMIM]benzoateg
Dark brown solution, analysis not possible
Dark brown solution, analysis not possible
a BMIM: 1-butyl-3-methylimidazolium. b NMIM: 1-methyl-3-nonylimidazolium. c HMIM: 1-hexyl-3-methylimidazolium. d OMIM: 1-methyl-3-octylimi-
dazolium. e 4-MBP: N-butyl-4-methylpyridinium. f MMIM: 1,3-dimethylimidazolium. g EMIM: 1-ethyl-3-methylimidazolium. h L-2 Candida antarctica
lipase B. i L-3 Candida rugosa lipase. j L-5 Candida antarctica lipase A. k L-6 Pseudomonas sp. lipase. l L-7 Pig pancreas lipase. m L-8 Thermomyces
lanuginosa lipase. n L-9 Mucor miehei lipase. o L-10 Alcaligenes sp. lipase.
2 G. Carrea and S. Riva, Angew. Chem., Int. Ed., 2000, 112, 2312.
In a typical experiment 1 mg of lipase is added to 400 ml
3 K. Faber, Biotransformations in Organic Chemistry, Springer, Berlin,
substrate solution containing 54 ml rac-6 and 122 ml 7 in 4.4 ml
2000.
ionic liquid or MTBE. The suspension is incubated for 3 d at 24
4 U. T. Bornscheuer and R. J. Kazlauskas, Hydrolases in Organic
Synthesis, Wiley-VCH, Weinheim, 1999.
5 J. M. S. Cabral, M. R. Aires-Barros, H. Pinheiro and D. M. F. Prazeres,
J. Biotechnol., 1997, 59, 133.
°C in a thermomixer. For analysis 100 ml of the reaction mixture
are extracted with 1 ml n-hexane–propan-2-ol (97.5+2.5). The
extract is analysed by HPLC using a chiral stationary Phase
Chiracel OJ (Daicel). The eluent consists of 96.5% (v/v) n-
hexane, 3% (v/v) propan-2-ol and 0.5% (v/v) ethanol with a
flow rate of 1 ml min21; temperature 38 °C, UV-detection at
205 nm.
6 A. M. Blinkorsky, Y. L. Khmelnitzky and J. S. Dordick, J. Am. Chem.
Soc., 1999, 116, 2697
7 B. Orlich and R. Schomäcker, Biotechnol. Bioeng., 1999, 65, 357.
8 T. Hartmann, E. Schwabe and T. Scheper, in Stereoselective Bio-
catalysis, ed. R. Pathel, Marcel Dekker, 2000, 799.
9 P. Wasserscheid and W. Keim, Angew. Chem., Int. Ed., 2000, 112,
3926.
10 K. R. Seddon, J. Chem. Tech. Biotechnol., 1997, 68, 351.
11 T. Welton, Chem. Rev., 1999, 99, 2071.
12 D. K. Magnuson, J. W. Bodley and D. F. Evans, J. Solution Chem.,
1984, 13, 583.
13 M. Erbeldinger, A. J. Mesiano and A. J. Russel, Biotechnol. Prog.,
2000, 16, 1129.
14 R. Madeira Lau, F. van Rantwijk, K. R. Seddon and R. A. Sheldon, Org.
Lett., 2000, 2, 4189.
For the distillative workup 600 mg lipase L-2 (10 U mg21) is
mixed with 4 ml [BMIM](CF3SO2)2N, 0.7 ml 6 and 1.2 ml 7,
mixed thoroughly and incubated at 40 °C for 40 min. Non-
converted starting material and (R)-8 are removed by vacuum
distillation at 85 °C and 0.06 mbar. After cooling down the same
amount of substrates is added again and the cycle is repeated.
We thank Roche Diagnostics GmbH, Mannheim, for provid-
ing the Chirazyme Screening set and additional amount of CAL,
and the Fonds der Chemischen Industrie for financial support.
15 S. G. Cull, J. D. Holbrey, V. Vargas-Mora, K. R. Seddon and G. J. Lye,
Biotechnol. Bioeng., 2000, 69, 227.
16 N. Kaftzik, P. Wasserscheid and U. Kragl, Angew. Chem., Int. Ed.,
submitted.
17 Ionic liquids are commercially available from Solvent Innovation
GmbH, Cologne (http://www.solvent-innovation.de).
18 A. M. Klibanov, CHEMTECH, 1986, 16, 354.
Notes and references
1 A. Liese, K. Seelbach and C. Wandrey, Industrial Biotransformations,
Wiley-VCH, Weinheim, 2000.
426
Chem. Commun., 2001, 425–426