Immobilised Burkholderia cepacia lipase in dry organic solvents and ionic liquids: A comparison

Article abstract of DOI:10.1039/b814606c

Lipase PS from Burkholderia cepacia in its free, commercial form (BCL-PS), immobilised in a sol-gel (BCLxero) and as a CLEA (BCL-CLEA) was tested in dry organic solvents, ionic liquids and their mixtures. Utilising the acylations of secondary alcohols 1-3 the influence of the enzyme preparation on its activity and enantioselectivity was studied. BCL-CLEA displays higher activity (initial rates) than BCLxero for all substrates in the ILs but loses its activity rapidly. Thus, BCLxero is suitable for kinetic resolution in ILs and in their mixtures with organic solvents. It is not possible to label one IL better than the other without taking the nature of the substrate into account. In neat solvents, the nature of the solvent affects enantioselectivity (E) only when furyl-substituted alcohol 2 serves as a substrate while variation in E is more evident for reactions in solvent mixtures.

Full text of DOI:10.1039/b814606c

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www.rsc.org/greenchem | Green Chemistry
Immobilised Burkholderia cepacia lipase in dry organic solvents and
ionic liquids: A comparison
Piia Hara,a Ulf Hanefeldb and Liisa T. Kanerva*a
Received 22nd August 2008, Accepted 7th November 2008
First published as an Advance Article on the web 4th December 2008
DOI: 10.1039/b814606c
Lipase PS from Burkholderia cepacia in its free, commercial form (BCL-PS), immobilised in a
sol–gel (BCLxero) and as a CLEA (BCL-CLEA) was tested in dry organic solvents, ionic liquids
and their mixtures. Utilising the acylations of secondary alcohols 1–3 the influence of the enzyme
preparation on its activity and enantioselectivity was studied. BCL-CLEA displays higher activity
(initial rates) than BCLxero for all substrates in the ILs but loses its activity rapidly. Thus,
BCLxero is suitable for kinetic resolution in ILs and in their mixtures with organic solvents. It is
not possible to label one IL better than the other without taking the nature of the substrate into
account. In neat solvents, the nature of the solvent affects enantioselectivity (E) only when
furyl-substituted alcohol 2 serves as a substrate while variation in E is more evident for reactions
in solvent mixtures.
alcohols while their nucleophilicities are much lower and entirely
Introduction
anion dependent, i.e., the ILs are unique as solvents showing
Ionic liquids (IL) and enzymes, as well as their application,
high polarity and low nucleophilicity.24–26 The cations are both
were both described long ago but it has taken until recently
modestly hydrophilic. Although it is well-known that [BF4] and
before the potential of combining them was recognised. They
[PF6] can release HF this is not the case when they are used
have been thoroughly reviewed as separate subjects and in
under dry conditions, as described here.9,16,25 Both [EMIM] and
combination.1–10 Using enzymes in ionic liquids many new
[BMIM] were recently assessed for their ecotoxicity and fall into
applications of biocatalysis have came about. However, to fully
a similar range as most organic solvents.27,28 The advantage of
appreciate the possibilities of applying enzymes in ionic liquids,
using these ILs lies therefore mainly in the fact that they are
the parameters influencing the behaviour of enzymes in ILs need
non-volatile and that a lot of previous work has been performed
to be understood. One of the parameters which has, to date,
in them. This allows a systematic comparison of the different
virtually not been systematically investigated, is the influence of
the enzyme preparation on its stability in different dry ILs or
IL/organic solvent mixtures.5
immobilisation techniques.
As test reactions, the kinetic resolution of aromatic alcohol 1,
heteroaromatic alcohol 2 and N-acylated amino alcohol 3 were
investigated (Scheme 1). In addition, the potential side reaction,
One of the enzymes best studied in ILs is Burkholderia cepacia
lipase (BCL), also known as Pseudomonas cepacia lipase and
hydrolysis of the acetate products 4–6 due to residual water in
lipase PS or lipase PS “Amano” SD.11–20 Here we report on
the enzymatic reaction mixture, was explored. This side reaction
the application of three different BCL forms, the commercial
causes the release of acetic acid, reduces the yield, affects
free enzyme powder from Amano which is sold diluted with
enantiopurities and is normally neglected. The significance of
dextrin, a cryoprotector,21 BCL encapsulated inside a sol–gel22
this side reaction is often ignored although ample evidence of
and BCL cross-linked as a CLEA.23 These forms of BCL are
its importance is available.29–34 Esterification of the acid was
utilised in different, often used ILs and in IL/organic solvent
previously proposed as the continuous source of water.35
mixtures for the enantioselective acylation of diverse racemic
In an earlier study we could already demonstrate that there
alcohols. A water miscible ionic liquid (IL) with a hydrophilic
is a significant difference in behaviour between the BCL prepa-
anion [EMIM][BF4] and water immiscible ionic liquids with
rations, and that depending on the substrate (1–3), different
hydrophobic anions [EMIM][Tf2N] and [BMIM][PF6] were
organic solvents should be employed.36 The commercial BCL
chosen. These ILs are thoroughly characterized (Table 1),
(BCL-PS) is in essence dextrin, containing 3% free enzyme. The
and display hydrogen bond donor abilities similar to those of
encapsulated enzyme is prepared with the sol–gel technique,
utilising both MTMS (methyltrimethoxysilane) and TMOS
(tetramethoxysilane) as precursors for the sol–gel.36,37 The
aDepartment of Pharmacology, Drug Development and
initially obtained aquagel is lyophilised to obtain the xerogel
which is then ready to be used as a catalyst. The final preparation
(BCLxero) is the enzyme encapsulated in reverse phase silica
in which part of the capsules will be broken due to the
drying process. The cross linked BCL (BCL-CLEA) is prepared
Therapeutics/Laboratory of Synthetic Drug Chemistry and Department
of Chemistry, University of Turku, Lemminka¨isenkatu 5 C, FIN-20520,
Turku, Finland. E-mail: lkanerva@utu.fi; Fax: +358 2 333 7955
bGebouw voor Scheikunde, Afdeling Biotechnologie, Technische
Universiteit Delft, 2628, BL, Delft, The Netherlands. E-mail:
u.hanefeld@tudelft.nl; Fax: +31 15 278 1415
250 | Green Chem., 2009, 11, 250–256
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Table 1 Properties of dry ILs: log P (logarithm for the partition coefficient of an IL between 1-octanol and water), EN (Reichardt’s normalized
T
polarity scale) and viscosity (25 ◦C)
24
Ionic liquid
log P24
EN
Viscosity [cP]25
Water content [ppm]a
Water solubility26
T
[EMIM][BF4]
[EMIM][Tf2N]
[BMIM][PF6]
Toluene
DIPE
TBME
-3.53
-1.18
-2.06
2.8
1.9
1.35
0.697
0.661
0.670
—
0.546
—
43
28
450
—
—
—
986
45
486
77
321
622
yes
no
no
no
no
no
a Determined by Karl Fisher titration.
Scheme 1 Kinetic resolution of three different alcohols catalysed by different BCL preparations, performed in organic solvents, ILs and IL/organic
solvent mixtures.
according to standard methodology.36,38 While the BCLxero had
the same or lower activity than BCL-PS in organic solvents the
Results and discussion
Racemates 1–3 were first subjected to acylation with vinyl acetate
in toluene, DIPE and TBME as well as in [EMIM][Tf2N],
[EMIM][BF4] and [BMIM][PF6] in the presence of the BCLxero
and BCL-CLEA (Table 2). Toluene for 1, DIPE for 2 and TBME
for 3 were previously best detected for the acylation in the
presence of BCLxero and BCL-CLEA.36 On the other hand,
the ILs were chosen as best for the previous acylation of 3 with
lipase PS-C II (BCL preparation on Toyonite from Amano16).39
Activities in the table are given as initial rates measured by
BCL-CLEA had higher activity (Tables 2 and 3).36 On the other
hand BCLxero proved more stable in organic solvents than BCL-
CLEA. We here describe the behaviour of the BCL preparations
in three different ILs and in their mixtures with selected organic
cosolvents with three different substrates (1–3). To make direct
comparison of the results possible, the protein content was
kept as 100 mg ml-1 BCLxero and BCL-PS, on one hand,
and as 50 mg ml-1 for BCL-CLEA and BCL-PS, on the other
hand.
Table 2 Acylation of 1–3 (0.1 M) with vinyl acetate (0.2 M) in organic solvents and in ionic liquids in the presence of BCLxero and BCL-CLEA at
room temperature
BCLxeroa
BCL-CLEAb
c
c
Entry
Substrate
Solvent
v0
Conv. [%]d/E
v0
Conv. [%]d/E
1
2
3
4
5
6
7
8
9
10
11
12
13
1
1
1
1
1
2
2
2
2
3
3
3
3
Toluene
Toluene
1.42 0.02
—
50/>200
—
30/>200
23/>200
34/>200
51/131
28/38
23/46
41/78
49/>200
5/>200
5/>200
13/>200
7.0 0.9
3.86 0.25e
0.32 0.01
1.65 0.07
1.1 0.1
30.1 0.4
0.17 0.01
2.28 0.25
1.34 0.38
6.7 0.3
50/>200
46/>200
5/>200
24/>200
18/>200
53/58
3/—
34/40
21/49
[EMIM][Tf2N]
[EMIM][BF4]
[BMIM][PF6]
DIPE
[EMIM][Tf2N]
[EMIM][BF4]
[BMIM][PF6]
TBME
0.31 0.01
0.19 0.01
0.41 0.01
1.47 0.02
0.90 0.01
0.15 0.02
0.27 0.04
1.54 0.03
0.03 0.01
0.04 0.01
0.08 0.01
49/>200
1/>200
17/—
[EMIM][Tf2N]
[EMIM][BF4]
[BMIM][PF6]
0.06 0.01
1.91 0.01
0.67 0.07
10/>200
a Based on 100 mg of BCL-PS powder. b Based on 50 mg of BCL-PS powder. c mmol min-1 g-1. d Conversion after 24 h/E. e CLEA prepared by adding
bovine serum albumin.
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Table 3 Acylation of 1–3 (0.1 M) with vinyl acetate in organic solvents and the mixtures of IL:organic solvent (1:2) in the presence of BCL-PS at
room temperature
Entry
Substrate
Solvent
Phases
BCL-PS
t [h]
c [%]
E
v0 [mmol min-1 g-1]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
1
1
1
2
2
2
2
3
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
Toluene
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
50 mg a
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
100 mg b
24
48
24
48
24
48
24
24
48
48
48
48
24
48
24
48
24
48
6
49
17
47
49
51
30
51
50
33
23
48
24
50
17
50
50
50
45
50
47
25
33
16
30
>200
>200
>200
>200
131
51
134
77
14
3.4 0.2
0.13 0.01
1.15 0.01
0.79 0.01
3.8 0.2
0.26 0.004
1.27 0.01
0.79 0.01
2.38 0.03
0.84 0.001
0.79 0.001
0.16 0.004
2.7 0.3
0.084 0.002
0.57 0.001
0.46 0.01
3.5 0.2
0.21 0.001
1.54 0.01
0.43 0.001
1.69 0.05
0.41 0.01
0.067 0.001
0.12 0.003
[EMIM][NTf2]:toluene
[EMIM][BF4]:toluene
[BMIM][PF6]:toluene
DIPE
[EMIM][NTf2]:DIPE
[EMIM][BF4]:DIPE
[BMIM][PF6]:DIPE
TBME
[EMIM][BF4]:TBME
[BMIM][PF6]:TBME
[EMIM][NTf2]:TBME
Toluene
[EMIM][NTf2]:toluene
[EMIM][BF4]:toluene
[BMIM][PF6]:toluene
DIPE
[EMIM][NTf2]:DIPE
[EMIM][BF4]:DIPE
[BMIM][PF6]:DIPE
TBME
[EMIM][BF4]:TBME
[BMIM][PF6]:TBME
[EMIM][NTf2]:TBME
50
70
>200
>200
51
>200
>200
115
88
115
196
30
24
24
24
48
48
50
15
30
a Corresponds to protein in BCL-CLEA. b Corresponds to protein in BCLxero.
mmol min-1 g-1 of the original BCL-PS used to prepare the xero
gel or the CLEA, thus allowing direct comparison. The results
clearly indicate that initial rates in the organic solvents (entries
1, 6 and 10) are always higher than those in the studied ILs.
The acylation in organic solvents proceeds smoothly close to
50% conversion in a highly enantioselective manner while in
the ILs there is a tendency for the progress of the acylation
to cease. Finally, the acylation of 3 in the ILs with both BCL
preparations practically stops at early stages (entries 11–13). The
BCL-CLEA displays higher initial rates than the BCLxero for all
substrates in [EMIM][BF4] and in [BMIM][PF6] (entries 4, 5, 8,
9, 12 and 13). Diffusion limitations can be proposed as a reason
for apparent low activities of the BCLxero. However, diffusion
limitation should be most evident in the viscose [EMIM][Tf2N]
rather than in [EMIM][BF4] or [BMIM][PF6] (Table 1). This is
not the case ruling out the influence of diffusion on the reaction
rates of BCLxero (Table 2, compare entries 3, 4, 5 and 7, 8, 9).
It can therefore be concluded that all three ILs have a negative
effect on the initial activity of BCLxero. For the more active
BCL-CLEA the diffusion limitations do become evident in the
same examples explaining the relatively low initial rate of BCL-
CLEA in [EMIM][Tf2N]. But as with BCLxero all ILs seem to
have a negative influence on the initial rates of BCL-CLEA. Poor
reactivity was reported earlier for the BCL-PS preparation in
ILs.12
Table 2). This loss of activity might be due to the earlier described
acetaldehyde-oligomer formation induced by ILs when vinyl
acetate is used as an acyl donor.17,40 Free BCL-PS was also
previously shown to loose its activity in [BMIM][PF6] and other
N,N-dialkyl imidazolium ion based ILs.12 The stabilisation of
BCL via immobilisation on the ceramic Toyonite carriers for
the use in ILs has earlier been described.16,17,39 The results
described here indicate that the xerogel can also protect BCL
against deactivation, while cross-linking does not provide this
stabilisation. Consequently BCLxero is the BCL preparation of
choice in IL.
No clear trend as to which IL is the best can be deduced from
Table 2. It seems that, much like the organic solvents, there is a
“best IL” for each of the three substrates. Thus it can be said
that with the organic solvent of choice for each substrate the
enzyme preparations always perform better than with the IL of
choice.
For kinetic resolutions of racemates, enantioselectivity (mea-
sured by the enantiomer ratio E) is often a more important
parameter than reactivity. In the case of 2, considerable solvent
effects on enantioselectivity are evident when either BCLxero
or BCL-CLEA was used (entries 6–9 Table 2). As for the
activity, the enzyme enantioselectivity is highly dependent on
the substrate. For this substrate [BMIM][PF6] is the IL of choice.
The excellent enantioselectivity for the acylations of 1 and 3 was
not affected by the solvents used (entries 1–5 and 10–13, Table 2).
In our previous work for the acylation of 3 with lipase PS–C
II, activities and enantioselectivities were enhanced when TBME
was added to [EMIM][Tf2N] and [EMIM][BF4].39 Accordingly,
toluene, DIPE and TBME were mixed with the ILs studied here,
and the acylation of 1–3 in the respective mixtures was investi-
gated using all three BCL preparations, BCL-PS, BCLxero and
While the initial rates of BCLxero are significantly lower
than those of BCL-CLEA the overall conversion in the IL with
BCLxero is much better for the acylation of 1 and 2. Indeed,
with BCLxero conversions continue to increase toward 50%
conversion with time. In contrast BCL-CLEA seems to loose
its activity rather rapidly and even with high initial rates the
reactions tend to stop at low conversions (entries 3, 4, 8, 9, 12, 13,
252 | Green Chem., 2009, 11, 250–256
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Table 4 Acylation of 1–3 (0.1 M) with vinyl acetate (0.2M) in the mixture of IL:organic solvent in the presence of BCLxero (based on 100 mg of the
original BCL-PS powder) at room temperature
Entry
Substrate
Solvent system
Phases
Conv.a [%]
eeb [%]/E
v0 [mmol min-1 g-1]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1
[EMIM][Tf2N]:toluene (2:1)
[EMIM][Tf2N]:toluene (1:1)
[EMIM][Tf2N]:toluene (1:2)
[EMIM][Tf2N]:toluene (1:3)
[EMIM][BF4]:toluene (2:1)
[EMIM][BF4]:toluene (1:1)
[EMIM][BF4]:toluene (1:2)
[EMIM][BF4]:toluene (1:3)
[BMIM][PF6]:toluene (2:1)
[BMIM][PF6]:toluene (1:2)
[EMIM][Tf2N]:DIPE (1:2)
[EMIM][BF4]:DIPE (2:1)
[EMIM][BF4]:DIPE (1:2)
[BMIM][PF6]:DIPE (2:1)
[BMIM][PF6]:DIPE (1:2)
[EMIM][Tf2N]:TBME (2:1)
[EMIM][Tf2N]:TBME (1:2)
[EMIM][BF4]:TBME (2:1)
[EMIM][BF4]:TBME (1:2)
[BMIM][PF6]:TBME (2:1)
[BMIM][PF6]:TBME (1:2)
1
2
2
2
1
2
2
2
1
2
2
2
2
2
2
1
2
1
2
1
2
40/49
22/34
45/49
—/30
33/45
43/48
39/47
29/40
37/47
46/50
51/54
51/—
54/—
51/—
34/33
10/18
26/37
32/42
28/37
5/5
98/>200
>99/>200
>99/>200
>99/>200
99/>200
99/>200
99/>200
>99/>200
99/>200
99/>200
85/66
91/66
87/61
87/42
96/60
>99/>200
97/126
96/71
96/89
>99/—
97/102
0.24 0.05
0.14 0.001
0.98 0.08
0.25 0.001
0.40 0.02
0.60 0.12
0.35 0.03
0.28 0.001
0.84 0.01
0.72 0.05
0.71 0.04
1.19 0.14
2.19 0.09
1.48 0.16
0.68 0.04
0.12 0.01
0.31 0.01
0.46 0.01
0.30 0.02
0.15 0.04
1.06 0.11
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3c
3c
3c
3c
3c
3c
41/44
a Conversion after 24 h/48 h. b ee for the formed ester product at the conversion after 48 h. c Reaction temperature 48 ◦C.
Table 5 Acylation of 1–3 (0.1 M) with vinyl acetate (0.2 M) in the mixture of an IL and an organic solvent in the presence of BCL-CLEA (based
on 50 mg of the original BCL-PS powder) at room temperature
Entry
Substrate
Solvent system
Phases
Conv.a [%]
eeb [%]/E
v0 [mmol min-1 g-1]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
[EMIM][Tf2N]:toluene (2:1)
[EMIM][Tf2N]:toluene (1:2)
[EMIM][BF4]:toluene (2:1)
[EMIM][BF4]:toluene (1:2)
[EMIM][BF4]:toluene (1:2)
[BMIM][PF6]:toluene (2:1)
[BMIM][PF6]:toluene (1:2)
[EMIM][Tf2N]:DIPE (1:2)
[EMIM][BF4]:DIPE (2:1)
[EMIM][BF4]:DIPE (1:2)
[BMIM][PF6]:DIPE (2:1)
[BMIM][PF6]:DIPE (1:2)
[EMIM][Tf2N]:TBME (1:2)
[EMIM][BF4]:TBME (2:1)
[EMIM][BF4]:TBME (1:2)
[BMIM][PF6]:TBME (2:1)
[BMIM][PF6]:TBME (1:2)
1
2
1
2
2
1
2
2
2
2
2
2
2
1
2
1
2
11/19
10/18
41/49
41/48
32/43
15/28
20/32
19/31
34/46
40/50
18/36
33/47
4/7
>99/>200
>99/>200
98/>200
>99/>200
>99/>200
>99/>200
>99/>200
95/49
92/57
93/79
96/79
94/78
>99/—
95/43
95/55
95/73
96/105
1.21 0.62
1.57 0.47
5.83 1.44
6.60 0.71
1.98 0.36
1.80 0.36
1.90 0.41
1.80 0.18
3.57 0.26
4.82 0.15
0.54 0.13
2.57 0.42
0.35 0.04
0.54 0.13
1.69 0.40
3.14 0.38
3.29 0.38
1
1
1
1c
1
1
2
2
2
2
2
3d
3d
3d
3d
3d
19/29
22/33
29/40
31/41
a Conversion after 24 h/48 h. b ee for the formed ester product at the conversion after 48 h. c CLEA prepared by adding bovine serum albumin.
d Reaction temperature 48 ◦C.
BCL-CLEA (Tables 3–5). The addition of an organic solvent
lowers the viscosity of an IL.41 Another important factor is that
the organic solvents used are not in all proportions soluble in
the ILs and separation into two phases may take place as given
in the tables.
All BCL preparations display the highest initial rates in the
pure organic solvent. Mixtures of ILs with organic solvents slow
down the reaction in most cases, independent of the fact whether
it is a mono- or biphasic mixture.
For substrate 1 BCL-PS clearly performs best in toluene, and
only in [EMIM][BF4]:toluene 1:2 mixture equal enantioselec-
tivity and close to 50% conversions after 24 h were observed
(Table 3, entries 1, 3, 13, 15). In IL organic solvent mixtures
BCLxero always displays lower activities for this substrate than
in toluene, but keeps excellent enantioselectivity (Table 2 entry 1
and Table 4 entries 1–10). For BCL-CLEA the activity towards
1 also uniformly drops in IL organic solvent mixtures compared
to the pure organic solvent, while excellent enantioselectivities
are maintained (Table 2 entry 1 and Table 5 entries 1–7).
For substrate 2 BCL-PS again performs best in the organic
solvent and in [EMIM][BF4]:DIPE 1:2 mixture while in the
other IL:DIPE mixtures lower activity and enantioselectivity are
observed (Table 3). In contrast BCLxero displays practically the
same (51–54%) conversion toward 2, independent of the solvent
system (Table 4, entries 11–14), the mixture [BMIM][PF6]:DIPE
(1:2) being an exception (entry 15). However, the
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enantioselectivity in IL:DIPE mixtures is always lower
than in pure DIPE (Table 2 entry 6 and Table 4 entries 11–15).
BCL-CLEA is less active and less enantioselective toward 2
when used in IL:DIPE mixtures compared to its use in pure
DIPE (Table 2 entry 6 and Table 5 entries 8–12).
and BCLxero proceeded smoothly without clear indications
about hydrolysis of the produced (S)-6 (Table 2, entry 10), the
hydrolysis in [EMIM][BF4] (1:2) (Table 4, entry 19) evidently
caused the observed difficulties in leading the acylation reaction
to 50% conversion and variations in E values with time.
In the case of substrate 3, BCLxero and BCL-CLEA have
the highest activity and enantioselectivity in TBME (compare
Table 2 entry 10 with Tables 4 and 5). This is not the case for
BCL-PS. Here improved enantioselectivity can be observed in
some cases in IL:TBME mixtures (Table 3, entries 9–12 and
21–24). In this case hydrolysis by the water in the enzyme may
complicate the interpretation of the observed results as will be
discussed below. However, the initial rates are always highest in
the pure organic solvent.
Thus, no clear trend-line can be deduced as to which reaction
medium should be used, mono- or biphasic. Instead it seems
that for each substrate the best solvent (mixture) has to be found
separately.
We then studied the hydrolysis of the produced enantiopure
esters (R)-4, (R)-5 and (S)-6 with the residual water in the system
with the two most successful BCL preparations, BCL-PS and
BCLxero. The potential of hydrolysis for the produced ester in
the acylation of 1–3 can be seen in such experiments while the
real proportion of the hydrolysis during the acylation cannot
be determined. Moreover, the proportion in the acylation is
certainly much less that the data in Table 6 suggest because
the concentration of the hydrolysable ester is initially zero
and the substrate alcohol (1–3) then competes with water as
a nucleophile. In the case of all the substrates, the presence
of BCLxero causes considerably less hydrolysis than BCL-PS
(Table 6). The hydrolysis of (S)-6 in the solvent mixture is
an exception (entry 6). In this case, the hydrolysable ester is
butanoate rather than acetate and the reaction takes place at
an elevated temperature. Another observation is that hydrolysis
is always more significant in the solvent mixture than in a neat
organic solvent. This might be explained by the relatively high
water content (986 ppm) of [EMIM][BF4] and by its water
miscibility. However, the same amount of water from the IL
is present in all the solvent mixtures (entries 2, 4 and 6), and
accordingly the water contents of the organic solvents (77 ppm
for toluene, 321 ppm for DIPE and 622 ppm for TBME)
make the difference. BCLxero in toluene and in the solvent
mixture caused practically no hydrolysis in the case of (R)-4
(entries 1 and 2) while considerable hydrolysis by the residue
water was observed in the case of (S)-6 in the solvent mixture
(entry 6). While the acylation of 3 in TBME with vinyl acetate
Conclusions
The present work indicates that the nature of the BCL prepara-
tion (native BCL-PS, BCLxero and BCL-CLEA in the present
work) on its activity is one of the crucial variables when enzymes
are used to catalyse organic reactions in the presence of ILs.
When these enzyme preparations were applied to the acylation
of three different aromatic secondary alcohols (1–3) in the most
commonly used ionic liquids ([EMIM][Tf2N], [EMIM][BF4]
and [BMIM][PF6) in biocatalysis it was shown that the ILs
have a negative influence on the initial rates (activities) of
the enzyme preparations compared to the reactions in selected
organic solvents or in their mixtures with an IL. While BCL-
CLEA displays higher activity (initial rates) than BCLxero for
all substrates in the ILs it loses its activity rapidly. In organic
solvents and in the ILs, the nature of the solvent affects E only
with 2 serving as the substrate while E > 200 is evident with 1 and
3. This work reveals that it is not enough to consider activities
of the BCL preparations to find the one with highest stability
against an IL. Rather the overall conversion and possible side
reactions, the tendency for hydrolysis of the ester produced in
particular, needs to be taken into consideration. Contrary to
earlier results,42 it is not possible to label one IL better than the
other without taking the nature of the substrate and the ester
produced into account. Overall, the results show that BCLxero
is a good choice, especially in cases where the produced ester
is an activated ester and thus susceptible to hydrolysis as a side
reaction.
Experimental
Materials
Lipase PS “Amano” SD (BCL-PS, from Burkholderia cepacia)
was purchased from Amano Pharmaceuticals Co., Ltd (Nagoya,
Japan). 1-Phenylethanol (98%) and 2-amino-1-phenylethanol
(98%) were products of Aldrich and 1-(2-furyl)ethanol (>97%)
was from Fluka. Amide 3 was prepared by the reac-
tion of 2-amino-1-phenylethanol with butanoic anhydride
(0.95 eqv.). (R)-4, (R)- 5 and (S)-6 were available from our
Table 6 Conversion (%) after 24 h in the hydrolysis of enantiopure esters 4–6 (0.05 M) by the residual water in the enzyme preparation and the
solvent system at room temperature
Entry
Substrate
Solvent
log P
2.8
Conv. [%] by BCL-PSa
Conv. [%] by BCLxerob
1
2
3
4
5
6
(S)-4
(S)-4
(S)-5
(S)-5
(R)-6c
(R)-6c
Toluene
[EMIM][BF4]:toluene (1:2)
DIPE
[EMIM][BF4]:DIPE (1:2)
TBME
[EMIM][BF4]:TBME (1:2)
4
27
20
22
86
35
1
2
10
26
16
45
1.9
1.35
a 100 mg mL-1 of BCL-PS. b BCLxero based on 100 mg mL-1 of BCL-PS. c Butanoate instead of acetate; reaction temperature 48 ◦C.
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previous work.36 Methyltrimethoxysilane (MTMS, Aldrich,
>99%), tetramethoxysilane (TMOS, Fluka, >99%) and glu-
taraldehyde (Fluka, 25% in water) were used as supplied.
[EMIM][Tf2N], [EMIM][BF4] and [BMIM][PF6] were pre-
pared by the methods described in the literature.36,39 Ethyl
bromide (>99%), 1-methylimidazole (>99%), 1-chlorobutane
(>99.5%), LiNTf2 (>99%) and sodium fluoroborate (>98%)
were products of Fluka. HPF6 (60% in H2O) was from
Aldrich. Vinyl acetate and the solvents were of the highest
grade from Aldrich, J.T. Baker and Lab-Scan Ltd. Water
contents of the neat solvents were measured by Karl Fischer
titration and were: toluene 77 ppm, TBME 622 ppm, DIPE
321 ppm, [EMIM][Tf2N] 45 ppm, [EMIM][BF4] 986 ppm and
[BMIM][PF6] 486 ppm.
buffer and centrifugated. The pellet was washed two times with
the buffer (3 mL) and once with acetonitrile (3 mL). After
centrifugatio◦ n, the obtained CLEA was dried in vacuum and
stored at 4 C. When BSA was used, it was added in KH2PO4
buffer together with BCL-PS.
Enzymatic acylation
For enzymatic acylation, an organic solvent (1 mL) or the
mixture of ionic liquid, solvent (1 mL) and vinyl acetate (0.2 M)
were added to one of the lipase preparations (for BCLxero
corresbonds to 100 mg and for BCL-CLEA 50 mg of original
BCL-PS powder; with BCL-PS both 50 and 100 mg were used)
and the addition of a substrate (0.1 M) started the reaction.
The reactions were shaken at room temperature (23 ◦C) for the
reactions of 1 and 2 and at 48 ◦C for the reaction of 3.
Analysis
The progress of the reactions was followed by taking samples
(50 mL) at intervals and extracting the products into toluene,
TBME or DIPE accordingly to organic solvent in the reaction.
The samples were derivatized with propionic anhydride in
the presence of 4,4-dimethylaminopyridine (DMAP, 1% in
pyridine) to achieve a good baseline separation and analyzed
by GC equipped with Chrompack CP-Chirasil-DEX CB column
(25 m ¥ 0.25 mm) and Chrompack CP-Chirasil-L-valine column.
The determination of E was based on equation E = ln[(1 -
c)(1 - eeS)]/ln[(1 - c)(1 + eeS)] with c = eeS/(eeS + eeP) using
linear regression (E as the slope of the line ln[(1 - c)(1 - eeS)]
versus ln[(1 - c)(1 + eeS)]. The protein content of lipase PS-SD
powder was determined using bicinchoninic acid assay using
bovine serum albumin as the standard protein.
Acknowledgements
This work was supported by the Academy of Finland (grant
210263 to L.T. K.). P.H. is thankful for the grant COST-STSM-
D25-02032.
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