Ionic liquid-coated enzyme for biocatalysis in organic solvent

Article abstract of DOI:10.1021/jo026116q

Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent. An ionic liquid, [PPMIM]-[PF₆] (1, [PPMIM] = 1-(3′-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53°C, was synthesized in two steps from N-methylimidazole. The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53°C and then allowing the mixture to cool. A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity.

Full text of DOI:10.1021/jo026116q

SCHEME 1. Syn th esis of Ion ic Liqu id
Ion ic Liqu id -Coa ted En zym e for
Bioca ta lysis in Or ga n ic Solven t
J ae Kwan Lee and Mahn-J oo Kim*
Department of Chemistry, Division of Molecular and
Life Sciences, Pohang University of Science and Technology,
San 31 Hyojadong, Pohang, Kyungbuk 790-784, Korea
coated enzyme (ILCE) that is readily prepared and
exhibits markedly enhanced enantioselectivity and reli-
able stability.
mjkim@postech.ac.kr
Received J une 28, 2002
Recently, a few groups including us5 have reported that
ionic liquids6,7 have great potential as alternative reaction
media for biocatalysis and biotransformation. It was
observed that their use enhanced the selectivity of the
enzyme.5c,d It was also demonstrated that they are useful
as media for the enzymatic reaction of polar substrates,
which are difficult to dissolve in conventional organic
solvents.5f One of the interesting properties of ionic
liquids, we think, is their insolubility in water or organic
solvents, which led us to envisage that they might be
suitable as the coating materials for immobilizing bio-
catalysts. Particularly, we thought that room-tempera-
ture solid-phase ionic liquids, which become liquid at
elevated temperature, would be of great use for such a
purpose. To test our idea, a novel ionic liquid [PPMIM]-
[PF6] (1, [PPMIM] ) 1-(3′-phenylpropyl)-3-methylimida-
zolium) was synthesized in good yields via two steps from
N-methylimidazole (Scheme 1). It was observed that the
ionic liquid was solid at room temperature and became
liquid over 53 °C.
Abstr a ct: Ionic liquid-coated enzyme (ILCE) is described
as a useful catalyst for biocatalysis in organic solvent. An
ionic liquid, [PPMIM]-[PF6] (1, [PPMIM] ) 1-(3′-phenylpro-
pyl)-3-methylimidazolium), which is solid at room temper-
ature and becomes liquid above 53 °C, was synthesized in
two steps from N-methylimidazole. The coating of enzyme
was done by simply mixing commercially available enzyme
with 1 in the liquid phase above 53 °C and then allowing
the mixture to cool. A representative ILCE, prepared with
a
lipase from Pseudomonas cepacia, showed markedly
enhanced enantioselectivity without losing any significant
activity.
Nonaqueous biocatalysis provide a useful component
of methodology in organic synthesis.1 For example, lipase
catalysis in organic solvents is of great use for the
synthesis of optically active compounds such as chiral
alcohols, acids, and their esters.2 However, biocatalysis
in nonaqueous media often suffers from reduced activity,
selectivity, or stability of enzyme.3 To overcome these
limitations, many approaches have focused on the de-
velopment of more efficient enzymes by enzyme modifica-
tion, molecular imprinting, additive addition, or substrate
matching.4 Some recent examples include cross-linked
enzyme crystals4a,b and aggregates,4c ligand4d-g or inor-
ganic salt4h co-lyophilized enzymes, and enzyme-coated
microcrystals.4i Although these modified enzymes exhibit
better activity, selectivity, or stability, the procedures for
their preparations in most cases are rather complicated.
We herein wish to report for the first time an ionic liquid-
As a representative enzyme for the preparation of
ILCE, Pseudomonas cepacia lipase (PCL)8 was chosen
since it had been frequently used for biotransformations
in organic solvents. In a typical procedure for the
preparation of ILCE, solid 1 was converted to its liquid
phase by heating above 53 °C. To this liquid were added
enzyme powders (0.1 mass equiv) and the resulting
mixture was stirred with a glass rod to get a uniform
heterogeneous solution. The solution was then allowed
to cool to room temperature until the enzyme-ionic liquid
mixture solidified. The solid phase was broken down to
a small size of particles with a spatula. The small ILCE
particles were then used without any further treatment
in the next experiments for testing their activity and
selectivity.
(1) (a) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon: Oxford, UK, 1994. (b) Enzyme Catalysis in
Organic Synthesis; Drauz, K., Waldmann, H., Eds.; VCH: Weinheim,
Germany, 1995; Vols. I and II. (c) Faber, K. Biotransformations in
Organic Chemistry, 3rd ed.; Springer: Berlin, Germany, 1997. (d)
Bornscheuer, U. T.; Kazlauskas, R. J . Hydrolases in Organic Synthesis;
Wiley-VCH: Weiheim, Germany, 1999.
The enantioselectivity of ILCE was examined with the
transesterification reactions of secondary alcohols 2a -e
(2) (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, Scotland, 1996.
(5) (a) Erbeldinger, M.; Mesiano, A. J .; Russel, A. Biotechnol. Prog.
2000, 16, 1131. (b) Lau, R. M.; Van Rantwijk, F.; Seddon, K. R.;
Sheldon, R. A. Org. Lett. 2000, 2, 4189. (c) Kim, K. W.; Song, B.; Choi,
M. Y.; Kim, M.-J . Org. Lett. 2001, 3, 1507. (d) Itoh, T.; Akasaki, E.;
Kudo, K.; Shirakami, S. Chem. Lett. 2001, 262. (e) Schoefer, S. H.;
Kraftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001, 425.
(f) Park, S.; Kazlauskas, R. J . Org. Chem. 2001, 66, 8395.
(6) Reviews: (a) Seddon, K. R. J . Chem. Technol. Biotechnol. 1977,
68, 351. (b) Welton. T. Chem. Rev. 1999, 99, 2071. (c) Wasserscheid,
P.; Wilhelm, K. Angew. Chem., Int. Ed. 2000, 39, 3772.
(7) (a) Ford, W. T.; Hauri, R. J .; Halt, D. J . Org. Chem. 1973, 38,
3916. (b) Bolkan, S. A.; Yoke, J . T. Iorg. Chem. 1986, 25, 3587. (c)
Wilkes, J . S.; Zaworotko, M. J . J . Electrochem. Soc. 1997, 144, 3881.
(d) Larsen, A. S.; Holbrey, J . D.; Tham, F. S.; Reed, C. A. J . Am. Chem.
Soc. 2000, 122, 7264.
(8) This enzyme is available from some commercial suppliers such
as Fluka, Roche, and Amano. We used the one provided by Amano.
(3) Klibanov, M. Trends Biotechnol. 1997, 15, 97.
(4) (a) St. Clair, N. L.; Navia, M. A. J . Am. Chem. Soc. 1992, 114,
7314. (b) Lalonde, J . J .; Govardhan, C.; Khalaf, C.; Martinez, A. G.;
Visuri, K.; Margolin, A. J . Am. Chem. Soc. 1995, 117, 6845. (c) Cao,
L.; Rantwijk, F. V.; Seldon, R. A. Org. Lett. 2000, 2, 1361 (d) Russell,
A. J .; Klibanov, A. M. J . Biol. Chem. 1988, 263, 11624. (e) Sta¨hl, M.;
J eppsson-Wistrand, U.; Mansson, M. O.; Mosbach, K. J . Am. Chem.
Soc. 1991, 113, 9366. (f) Rich, J . O.; Dordick, J . S. J . Am. Chm. Soc.
1997, 119, 3245. (g) Ke, T.; Klibanov, A. M. Biotechnol. Bioeng. 1998,
57, 764. (h) Khmelnitzky, Y. L.; Welch, S. H.; Clark, D. S.; Dordick, J .
S. J . Am. Chem. Soc. 1994, 116, 2647. (i) Kreiner, M.; Moore, B. D.;
Parker, M. C. Chem. Commun. 2001, 12, 1096. (j) Lee, D.; Choi, Y. K.;
Kim, M.-J . Org. Lett. 2000, 2, 2553.
10.1021/jo026116q CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/20/2002
J . Org. Chem. 2002, 67, 6845-6847
6845

SCHEME 2. Lip a se-Ca ta lyzed Tr a n sester ifica tion
F IGURE 1. The relative activity of ILCE. One control reaction
with native enzyme and five ILCE-catalyzed reactions were
performed in the presence of 2a and vinyl acetate in toluene
at 25 °C for 1day. In the ILCE-catalyzed reactions, the first
run was carried out with fresh ILCE and the second to fifth
runs were done with recycled ILCE. The conversion percent
in each run was compared with that in the control reaction to
obtain the relative activity.
TABLE 1. Th e En a n tioselectivities in th e
Lip a se-Ca ta lyzed Tr a n sester ifica tion s of 2a -e in
Tolu en ea ,b
entry
substrate
lipase
ees
eep
E
2c, the enantioselectivity enhancement by the use of
ILCE was about 2-fold (see entries 1-2 and 4-5, respec-
tively). In the reactions of 2d -e, the enantioselectivity
enhancements by the use of ILCE were about 1.5-fold.
In the reaction of 2b, however, the enantioselectivities
in both cases were similar (compare entries 3 and 4).
Overall, these results indicate that the ionic liquid-coated
enzyme in most cases is more selective than its native
counterpart.
1
2
3
4
5
6
7
8
9
2a
native
ILCE
native
ILCE
native
ILCE
native
ILCE
native
ILCE
0.545
0.295
0.340
0.220
0.563
0.370
0.459
0.300
0.575
0.275
0.987
0.995
0.986
0.986
0.988
0.995
0.971
0.983
0.978
0.989
265
532
198
176
293
574
107
156
161
237
2b
2c
2d
2e
10
The catalytic efficiency and stability of ILCE were
examined with conversion percent in the transesterifi-
cation reaction of 2a , which was carried out in the
presence of vinyl acetate in toluene at 25 °C for 1 day.
The ILCE-catalyzed reactions were repeated five times
with the recycling of enzyme. In first run, the fresh ILCE
displayed 65% of the activity expected from native
enzyme used. However, the catalytic activity of ILCE
increased up to the level of native enzyme in the second
run and then was slightly reduced in the following runs.
After the fifth run, ILCE retained 93% of the activity of
its native counterpart (Figure 1). The reduced activities
of fresh ILCE in the first run seem to be due to some
diffusion difficulty, which should be relieved with a
decrease in its particle size as the reaction proceeds.
These observations indicate that lipase loses practically
no significant activity during the coating process and its
coated form has satisfactory stability.
In conclusion, this work has demonstrated that lipase
shows enhanced enantioselectivity without losing any
significant activity when it is coated with an ionic liquid
1. The ionic liquid is readily available and the coating
procedure is simple and straightforward. Furthermore,
the coated lipase is easy to reuse and retains its full
activities even after several runs. Accordingly, it is
believed that ILCE should find use as a new type of
immobilized biocatalyst for biotransformations in organic
solvents. Further studies to broaden the scope of ILCE
toward other enzymes and organic solvents are in progress
at this laboratory.
a
Experimental procedure: The ILCE-catalyzed reaction of 2a
is described as a representative procedure. 2a (15 mg, 0.1 mmol),
vinyl acetate (28 µL, 0.3 mmol), and ILCE (165 mg) were mixed
with toluene (0.5 mL), and the resulting heterogeneous mixture
was stirred at 25 °C for 1 day. The reaction mixture was then
filtered to remove enzymes, concentrated, and finally subjected
to silica gel chromatography to provide the unreacted substrate
(S)-2a (11 mg, 0.075 mmol, 75%, 29.5% ee) and the acetylated
b
product (R)-3a , (4 mg, 0.021 mmol, 21%, 99.5% ee). The optical
purities were determined by HPLC using a chiral column. Analyti-
cal conditions: Chiralcel OD, hexane/2-propanol 90/10 (2c), 95/5
(2a , 2d ), and 97/3 (2e), flow rate ) 1.0 mL/min (2a , 2c-e), UV 250
(2c), 217 nm (2a , 2d , 3e); Whelk-O1, hexane/2-propanol 99/1 (2b,
3a , 3c, 3b), 95/5 (3e), and 94/6 (3d ), flow rate ) 1.0 mL/min (2b,
3a -e), UV 250 (3c), 217 nm (2b, 3a ,b,d ,e).
in the presence of vinyl acetate in toluene at 25 °C
(Scheme 2). For comparison, the same reactions were
carried out with native enzyme. In typical experiments,9
an enzymatic reaction was performed with a solution
containing substrate (0.1 mmol), lipase (native, 15 mg;
ILCE, 165 mg), and vinyl acetate (3 equiv) in toluene (0.5
mL) at 25 °C for 1 day. The enzymes were then removed
by filtration and the resulting solution was concentrated.
The organic residue was subjected to silica gel chroma-
tography to obtain unreacted substrate and acetylated
product. Their optical purities (ees and eep) were then
determined by HPLC with use of a chiral column. The E
values were calculated by using the following equation,
E ) ln[1 - c(1 + eep)]/ln[1 - c(1 - eep)], where c ) ees/
(ees + eep).10 The results are given in Table 1.
The ILCE-catalyzed transesterifications of 2a -e except
one case proceeded with better enantioselectivity than
those catalyzed by native PCL. In the reactions of 2a and
Exp er im en ta l Section
P r ep a r a tion of Com p ou n d 1. 3-Phenylpropyl chloride (19
g, 0.125 mol) was dissolved in 1-methylimidazole (10 mL, 0.125
mol) and then reflux for 24 h at 70 °C. To the reaction mixture
was added acetone (100 mL) and NaPF6 (21 g, 0.125 mol) at 0
°C and then the resulting solution was stirred for 48 h at room
(9) See footnote a in Table 1 for the detailed experimental procedure.
(10) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294.
6846 J . Org. Chem., Vol. 67, No. 19, 2002

temperature. After filtering out precipitate, the organic layer
was dried and concentrated in vacuo to afford the desired
product (1, 45 g, 0.119 mol, 95%): mp 52-53 °C; 1H NMR (CD3-
CN, 300 MHz, ppm) 2.14 (m, 2H, CH2), 2.65 (t, 2H, J ) 7.4, CH2),
3.80 (s, 1H, NCH3), 4.14 (t, 2H, J ) 7.2, CH2), 7.19-7.35 (m,
7H), 8.34 (s, 1H, CH). Anal. Calcd for C13H17F6N2P: C, 45.09;
H, 4.95; N, 8.09. Found: C, 45.16; H, 4.94; N, 8.08.
P r ep a r a tion of ILCE. Solid 1 (1 g) was heated above 53 °C
in a flask to get a liquid phase, followed by the addition of lipase
(0.1 g). The resulting heterogeneous solution was stirred with a
glass rod for 1 min and then cooled to room temperature to yield
ILCE (1.1 g) in a solidified solution.
Ack n ow led gm en t. This work was supported by the
Korean Ministry of Science and Technology (the NRL
program, 2000-2002) and POSCO. We thank the Ko-
rean Ministry of Education for financial support to our
graduate program by the BK21 program.
J O026116Q
J . Org. Chem, Vol. 67, No. 19, 2002 6847