thermolysin-catalyzed synthesis of Z-aspartame in ionic
liquids containing 5% (v/v) water has been reported by
Erbeldinger et al.16 Herein we report the first example of
the use of a free enzyme in an ionic liquid in the absence of
water.
Table 1. Transesterification of Ethyl Butanoate with
Butan-1-ol Catalyzed by Free and Immobilized Candida
antarctica Lipase Ba
solvent
lipase
time (h)
conv (%)
Lipases tolerate nonnatural reaction conditions surprisingly
well and hence are ideal candidates for exploring the
possibilities of ionic liquids. Lipases, which in nature catalyze
the hydrolysis of triacyl glycerides, readily catalyze related
reactions such as esterification or transesterification in
anhydrous organic media.
[
[
C4mim][BF4]
C4mim][PF6]
CaLB (Nov 435)
CaLB (Nov 435)
CaLB (Nov 435)
CaLB (Nov 435)
CaLB (SP 525)
CaLB (SP 525)
4
4
4
4
24
24
81
81
74
66
79
78
t-BuOH
-BuOH
C4mim][BF4]
[C mim][PF ]
1
[
4
6
The ionic liquids 1-butyl-3-methylimidazolium tetrafluo-
a
Reaction conditions: 40 mM ethyl butanoate, 200 mM butan-1-ol, and
6
roborate ([C
4
mim][BF
4
]) or hexafluorophosphate ([C
4
mim]-
25 mg of enzyme in 1 mL of solvent were stirred at 40 °C.
12
[
6
PF ]) were used as solvent. The former salt was used
when the effects of water were to be studied, since this
compound is miscible with water in all proportions, whereas
the latter is hydrophobic.
The transesterification was also performed with free CaLB
Novo SP525) suspended in the ionic liquid. A slower
(
reaction was observed, compared with Novozym 435,
although the active protein content of SP525 is much higher.
This is consistent with the results obtained in organic media
where suspensions of “free” lipases are generally less
The transesterification of ethyl butanoate with butanol in
both salts of [C mim] was studied using Candida antarctica
4
lipase B (Novozym 435) as catalyst (Figure 2). The butyl
1
8
effective than immobilized lipases.
The transesterification of ethyl octanoate with butan-1-ol
and propan-2-ol was performed in the presence of Cal B
(Novozym 435) (Table 2). With butan-1-ol a higher conver-
Table 2. Transesterification of Ethyl Octanoate Using
Novozym 435a
solvent
alcohol
conv (%, 24 h)
[
C4mim][PF6]
butan-1-ol
butan-1-ol
propan-2-ol
propan-2-ol
81
69
56
81
t-BuOH
C4mim][PF6]
t-BuOH
[
Figure 2. Transesterification of ethyl butanoate with butan-1-ol
a
Reaction conditions: 10 mM ethyl octanoate, 120 mM alcohol, and
5 mg of Novozym 435 in 0.5 mL of solvent were stirred at 40 °C.
catalyzed by Novozym 435 in different ionic liquids: ([) [C
4
mim]-
2
[
BF ]; (9) [C mim][PF ]; (2) [C mim][BF ]/H O (9:1, v/v).
4
4
6
4
4
2
Reaction conditions: 40 mM ethyl butanoate, 200 mM butan-1-ol,
and 25 mg of enzyme in 1 mL of solvent were stirred at 40 °C.
sion was observed in [C
-ol the conversion was higher when tert-butyl alcohol was
used as solvent.
4 6
mim][PF ], whereas with propan-
2
ester was formed in more than 80% yield when the reaction
was carried out in an anhydrous ionic liquid (83% yield is
expected at equilibrium, on the basis of the molar ratio of
Primary fatty acid amides are important commodities that
can be prepared under mild conditions via a lipase-catalyzed
19
reaction of the carboxylic ester and ammonia. We compared
the ammoniolysis of ethyl octanoate in an ionic liquid
medium with that in tert-butyl alcohol under otherwise
identical reaction conditions (Table 3). The conversion into
ester and alcohol). The anion had only a small effect on the
-
6
reaction rate; the initial rate was slightly lower in the [PF ]
salt. In the presence of water (10% v/v), butanoic acid was
formed, as would be expected, in 62% yield, resulting in a
correspondingly lower yield of butyl ester.
4 4
octanamide was markedly lower in [C mim][BF ] than in
tert-butyl alcohol. The several preparations of Cal B (im-
For comparison, the reaction was also performed in tert-
butyl alcohol and butan-1-ol (Table 1). The transesterification
was slightly faster in the ionic liquids than in tert-butyl
alcohol. The somewhat lower reaction rate in butan-1-ol is
to be ascribed to inhibition by the solvent.17
18
mobilized on Lewatit E, free and immobilized on EP100 )
show different performances, the free enzyme showing the
lowest activity in this system (38% conversion after 24 h,
whereas Novozym 435 shows 40% and 57% for SP525 on
EP100).
(
15) Cull, S. G.; Holbrey, J. D.; Vargas-Mora, V.; Seddon, K. R.; Lye,
G. J. Biotechnol. Bioeng. 2000, 69, 227-233.
16) Erbeldinger, M.; Mesiano, A. J.; Russell, A. J. Biotechnol. Prog.
000, 16, 1131-1133.
17) De Zoete, M. Ph.D. Thesis, Delft University of Technology, Delft,
The Netherlands, 1995.
(18) Van Rantwijk, F.; Kock-van Dalen, A. C.; Sheldon, R. A. In Stability
and Stabilisation of Enzymes; Ballasteros, A., Plou, F. J., Iborra, P., Halling,
Eds.; Progress in Biotechnology, Elsevier Science: New York, 1998; Vol.
15, pp 447-452.
(19) De Zoete, M. C.; Kock-Van Dalen, A. C.; Van Rantwijk, F.;
Sheldon, R. A. Biocatalysis 1995, 10, 307-316.
(
2
(
4190
Org. Lett., Vol. 2, No. 26, 2000