Effective resolution of 1-phenyl ethanol by Candida antarctica lipase B catalysed acylation with vinyl acetate in protic ionic liquids (PILs)

Article abstract of DOI:10.1039/c2gc35196j

Herein we report the novel use of essentially anhydrous protic ionic liquids (PILs) as reaction media for performing the smooth kinetic resolution of 1-phenylethanol by Candida antarctica lipase B (CaLB) catalysed acylation with vinyl acetate at 40°C. Good rates and high enantioselectivities (92->99%) were observed with suspensions of both the free enzyme and cross-linked enzyme aggregates (CLEAs) of CaLB. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2gc35196j

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Effective resolution of 1-phenyl ethanol by Candida antarctica lipase B
catalysed acylation with vinyl acetate in protic ionic liquids (PILs)
Antonia P. de los Ríos,† Fred van Rantwijk and Roger A. Sheldon*
Received 9th February 2012, Accepted 20th March 2012
DOI: 10.1039/c2gc35196j
hydrolysis catalysed by a lipase dissolved in water and the corre-
sponding esterification catalysed by a suspension of the same
lipase in an organic solvent. One method for improving the
activity of enzymes in organic solvents is to lyophilize them in
the presence of added salts,5,6 such as potassium chloride, typi-
cally 94–98% of the total weight, first described by Khmelnitsky
and coworkers.5 This led us to the notion that suspension of an
enzyme in a room temperature ionic liquid (IL), with its salt and
water like character, could afford significant rate improvements
compared to organic solvents. Ionic liquids (ILs) are substances
that are composed entirely of ions and are liquid at or close to
room temperature. Interest in their use as reaction media7 in par-
ticular for catalytic processes,8 has increased exponentially over
the last two decades. This interest stems from their negligible
vapour pressure, thermal stability and widely tunable properties,
such as polarity, hydrophobicity and solvent miscibility behav-
iour, through manipulation of the cation and anion. Hence, they
have been widely advocated as green alternatives to volatile
organic solvents.
In our original investigation we showed that Candida antarc-
tica lipase B (CaLB) is able to catalyse a variety of reactions –
transesterification, amidation and perhydrolysis – as a suspension
in the dialkylimidazolium ILs, [bmim] [BF4] and [bmim] [PF6],
containing the non-coordinating tetrafluoroborate and hexafluoro-
phosphate anions.9 On the one hand, the results were very excit-
ing but on the other hand they were rather disappointing as rates
were only marginally higher than those observed in the best
organic solvents (e.g., tert-butyl alcohol, toluene and ethers).
Nonetheless, this and subsequent studies showed that anhydrous
ILs are eminently biocompatible with enzymes and can afford
stabilities that are substantially higher than in standard organic
solvents.10 A pertinent example is the reported esterification of
phthalic acids with ethanol catalysed by Bacillus thermocatenu-
latus lipase (BTL) in [bmim][BF4] at 120 °C.11 In some cases
higher enantioselectivities have been observed in ILs compared
to organic solvents.12
Herein we report the novel use of essentially anhydrous
protic ionic liquids (PILs) as reaction media for performing
the smooth kinetic resolution of 1-phenylethanol by Candida
antarctica lipase B (CaLB) catalysed acylation with vinyl
acetate at 40 °C. Good rates and high enantioselectivities
(92–>99%) were observed with suspensions of both the free
enzyme and cross-linked enzyme aggregates (CLEAs) of
CaLB.
Introduction
The seminal paper of Zaks and Klibanov1 on enzymatic catalysis
in organic media at 100 °C, published in 1984, was a watershed
in the use of enzymes in organic synthesis and heralded the
beginning of so-called “non-aqueous enzymology.”2 Many
organic substrates are only sparingly soluble in water and the
widely held notion that applications of enzymes were limited to
aqueous media constituted a formidable obstacle to their wide-
spread use in organic synthesis. Hence, the observation that bio-
catalysis was not only feasible in non-aqueous media but that
many enzymes were more thermally stable in organic solvents
than in water led, in the following decades, to the broad appli-
cation of biocatalysis as a mainstream synthetic tool. Today,
almost three decades later, the industrial scope of biocatalysis
continues to expand. Biocatalysis in non-aqueous media affords
many additional benefits.3 For example, certain reactions, such
as esterification and amidation, are difficult to perform in
aqueous media owing to equilibrium limitations and/or compet-
ing hydrolytic side reactions, but can be readily conducted in
organic solvents. Other benefits are easier product recovery from
low boiling solvents and elimination of microbial contamination.
However, a price has to be paid for these benefits and the
paradox of non-aqueous enzymology is that although enzymes
are able to perform as suspensions in organic solvents their cata-
lytic efficiencies are two or more orders of magnitude lower than
those observed in water.4 We note, however, that it is difficult to
make a meaningful comparison between the rate of an ester
Following this initial report in 2000, biocatalysis in anhydrous
ionic liquids has been widely studied and is the subject of
numerous reviews.13–25 It has led to the development of new
reaction concepts e.g., recycling of the biocatalyst with the ionic
liquid phase,26 the use of scCO2 as a mobile phase for the extrac-
tion of products from the IL in a continuous operation mode27
and the application of a scCO2–ionic liquid miscibility switch28
and the use of an ionic acylating reagent in enzymatic kinetic
resolution.29,30
Laboratory of Biocatalysis and Organic Chemistry, Department of
Biotechnology, Delft University of Technology, Julianalaan 136, 2628
BL Delft, The Netherlands. E-mail: r.a.sheldon@tudelft.nl
†Current address: Department of Chemical and Environmental Engi-
neering, Technical University of Cartagena, 30202, Cartagena, Spain.
1584 | Green Chem., 2012, 14, 1584–1588
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As noted above, the use of ionic liquid media for biotrans-
formations was stimulated by the possibility of replacing volatile
organic solvents with ILs. Owing to their negligible vapour
pressure the risk of air pollution is largely circumvented.
However, they do have significant solubility in water and, hence,
the environmental fate and ecotoxicity of ILs is a cause for
concern.31 The fact that they have high chemical and thermal
stabilities suggests that problems with poor biodegradability,
bioaccumulation and aquatic ecotoxicity can be expected.
Indeed, the first generation dialkylimidazolium ILs, such as
[bmim][BF4] and [bmim][PF6] and tetraalkylammonium ILs
exhibit acute toxicity towards a variety of aquatic organisms32–34
and are poorly biodegradable.35 Another drawback of these first
generation quaternary ILs is that they are prohibitively expensive
to make and not all of the synthetic methods are green.36 Conse-
quently, second generation ILs derived from more eco-friendly
natural products,37 such as carbohydrates38 and amino acids39
are being developed, preferably from inexpensive raw materials.
For example, 2-hydroxyethylammonium lactate, comprises a
cation closely resembling that of the natural cation, choline, and
a readily biodegradable anion.40
stability and high enantioselectivity.45 This is, to our knowledge,
the first report of biocatalysis in such PILs.
Results and discussion
We investigated the enantioselective acylation of 1-phenyletha-
nol with vinyl acetate (Fig. 1), a model reaction for alcohol resol-
ution, in the presence of CaLB. The kinetic resolution was
carried out at 40 °C in a number of PILs as well as in tert-butyl
alcohol as a reference.
Effects of the ionic liquid structure
Two types of ionic liquids were investigated: fatty acid salts of
trialkylamines (1–5, see Table 1) as well as similar ionic liquids
that contained a primary alcohol function (7–10). A dissolution
test showed that none of these ionic liquids solubilised CaLB.
The initial reaction rates that we observed in 1–5 ranged from
somewhat lower than in tert-butyl alcohol, as was the case with
ionic liquid 1, to four times as high (2 and 4, see Table 2). The
high enantioselectivities observed (see Table 2) were consistent
with negligible competition from non-enzymatic background
reactions, which one might have expected to arise from the pres-
ence of free amine resulting from dissociation of the PILs. This
was confirmed in blank experiments in the absence of enzyme.
In our search for inexpensive ionic liquids with reduced eco-
toxicity and better biodegradability, that are suitable for perform-
ing biotransformations, our attention was drawn to protic ionic
liquids (PILs) that contain
a protonated N-atom in the
cation.41–43 PILs are exquisitely simple to prepare by simply
mixing a tertiary amine with an acid, such as a carboxylic acid
and are known44 to exhibit better biodegradability and lower
toxicity than the corresponding quaternary ammonium salts.
Moreover, they have suitable H-bond donating properties for
interaction with and stabilization of enzymes and, when com-
bined with alkanoate anions, they are self-buffering. Herein we
describe the application of a range of PILs, prepared by simply
mixing tertiary amines and carboxylic acids, in the kinetic resol-
ution of 1-phenylethanol catalysed by Candida antarctica lipase
B (CaLB). The latter was used on account of its operational
Table 1 Ionic liquids used in this study
Ionic liquids [R1R2R3NH+][X−]
R1
R2
R3
X−
1
2
3
4
5
6
7
8
9
CH3
CH3
i-C4H9
CH3
n-C8H17
CH3
CH3
CH3
n-C4H9
CH3
CH3
i-C4H9
CH3
n-C8H17
n-C4H9
n-C4H9
i-C4H9
n-C12H25
n-C8H17
–(CH2)5–
CH2CH2OH
CH2CH2OH
CH2CH2OH
Propionate
3-Hexyldecanoate
Hexanoate
Octanoate
Acetate
Acetate
CH3
CH3
n-C4H9
Propionate
Decanoate
Octanoate
Ionic liquid [R1R2R3R4N+][X−]
R1
R2
R3
R4
X−
10
CH3
CH3
CH3
CH2CH2OH
propionate
Fig. 1 Kinetic resolution of 1-phenylethanol.
Table 2 CaLB-mediated resolution of 1-phenylethanol in ionic liquidsa
Initial rate
Initial rate
(nmol min−1 U−1
)
Time 50% conv. (d)
eeproduct (%)
Free
(μmol min−1 g−1
)
Medium
Free
CLEA
CLEA
Free
CLEA
Free
CLEA
1
2
3
4
14
2
2
1
4
7
8
4
1
2
7
95
99
93
99
>99
>99
97
95
92
99
>99
>99
5.8
30.7
27.0
46.1
13.8
10.6
5.7
72.8
20.4
48.1
24.9
17.2
0.16
0.85
0.75
1.28
0.38
0.29
0.55
7.0
2.0
4.6
2.4
1.6
5
t-BuOH
11
a Experimental details are in the Experimental section.
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Within this series no obvious relationship between activity
and ionic liquid structure became apparent, although it is worth
noting that relatively high activities were observed in the bulky
ionic liquids (2 and 4) compared with 1. Even the basic acetate
anion in 5 was tolerated rather well by CaLB. Neither did an
obvious correlation with the Kamlet–Taft solvent parameters (see
Table 3, Experimental section) become apparent. The time
course of the reactions in PILs 1–5 and tert-butyl alcohol are
depicted in Fig. 2.
The 1-methylpiperidinium-derived ionic liquid 6 proved an
exception to the above, as acylation in this latter solvent was >10
times slower than in tert-butyl alcohol. Its molecular size is only
slightly smaller than that of 1 (seven vs. nine C-atoms), however;
its mediocre performance may be due to a combination of a com-
paratively small cation (six C-atoms) and the basic acetate anion.
Transesterification in the hydroxylated ionic liquids 7–10 was, at
<4% per day, too slow to be of practical use and the enantio-
selectivity also was generally poor (data not shown).
exchanged positions in the order of relative rate but effects on
the enantioselectivity were minor.
Conclusions
Protic ionic liquids (PILs), prepared directly from simple tertiary
amines and carboxylic acids, were shown to be eminently suit-
able reaction media for conducting the enzymatic resolution of
1-phenyl ethanol, in good rates and excellent enantioselectivities.
These results, coupled with the ease of preparation of PILs from
inexpensive raw materials and their lower ecotoxicity and better
biodegradability compared with analogous quaternary ILs,
makes them attractive media for performing enzymatic processes
on a large scale. Consequently, we expect that PILs will find
broad application as reaction media for biotransformations in the
future.
Experimental
Effects of immobilisation
Materials
To assess the effects of immobilisation, a commercial preparation
of cross-linked enzyme aggregates (CLEAs) of CaLB46,47 was
compared with a suspension of free CaLB, which had been
obtained from the commercial, stabilised solution by precipi-
tation with acetone. Such a comparison is not straightforward,
however, as the preparations have different histories. Any salts
and other low molecular weight adjuvants present as stabilisers
in the commercial preparation remain in the suspended precipi-
tate, whereas they will be washed from the CLEA in the course
of its preparation.48 Hence, comparing the activities on the basis
of weight would make little sense. In Table 2 we compare trans-
esterification rates on the basis of hydrolytic activity, which
makes sense as it also takes any permanent loss of catalytically
competent active sites49 into account. When compared on the
basis of hydrolytic activity, the CLEA is a three to eight times
more efficient transesterification catalyst than the free enzyme, in
particular with ionic liquid 2. Presumably, the cross-linking
stabilizes the native, active conformation of CaLB in the anhy-
drous reaction media.
Candida antarctica lipase B (CaLB, Novozym 525L, batch no.
PPW 6354, 7.9 U mL−1) was obtained from Novozymes (Bags-
værd, Denmark). Cross-linked enzyme aggregates of Candida
antarctica lipase B (batch no. B07325, 10.4 kU g−1) were kindly
donated by CLEATechnologies (Delft, The Netherlands).
Trialkylammonium-based ionic liquids (see Table 1, purity
>95%; water <0.25%) were from Bioniqs Ltd (York, UK) and
were obtained as a gift from CLEA Technologies. Kamlet–Taft
parameters, insofar as known, are listed in Table 3. Prior to use
they were thoroughly dried by allowing them to stand for at least
24 hours at room temperature in a desiccator containing a very
large excess of phosphorus pentoxide compared to any residual
water. 1,2,3-Trimethoxybenzene and 1-phenyl ethanol were pur-
chased from Aldrich and vinyl acetate was from Acros Organics.
Zeolite KA (powder) was obtained from Aldrich and activated at
400 °C prior to use. HPLC grade tert-butyl methyl ether
(TBME) and tert-butyl alcohol were from Acros Organics and
Riedel-de Haën and were dried over activated zeolite NaA
(Aldrich) before use. All other reagents were of the highest
purity available.
Apart from the catalytic efficiency, immobilisation affected the
performance of CaLB only slightly. Ionic liquids 2 and 4
Table 3 Solvent parameters of ionic liquids 1–9
Solvent parametersa
Ionic liquid
α
β
π*
EN
T
1
2
3
4
5
6
7
8
9
1.42
1.61
0.95
0.83
0.98
0.83
0.66
0.50
0.40
0.53
0.53
0.69
0.85
0.72
0.74
0.92
0.95
1.43b
0.88b
0.85
0.92
0.99
0.92
0.90
0.87
1.34
1.27
a Solvent parameters have been taken from the Bioniqs solvent catalogue
2009 unless indicated otherwise. b Values taken from ref. 41.
Fig. 2 Reaction progress in the presence of free CaLB. Medium:
1 (□), 2 (4), 3 (◇), 4 (◯), 5 (●), tert-butyl alcohol (◆).
1586 | Green Chem., 2012, 14, 1584–1588
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Analysis and equipment
Acknowledgements
The enantioselective acylation of 1-phenylethanol was monitored
on a Shimadzu GC-17A instrument equipped with a FID detec-
tor and a Varian Inc. 25 m × 0.32 mm Chirasil-Dex CB column
at 120 °C (isothermal). Retention times: (S)-1-phenylethyl
acetate, 2.4 min; (R)-1-phenylethyl acetate, 2.6 min; (R)-1-phe-
nylethanol, 3.2 min; (S)-1-phenylethanol, 3.4 min; 1,2,3-tri-
methoxybenzene (internal standard), 4.0 min.
Donations of Candida antarctica lipase and ionic liquids by
CLEA Technologies (Delft, The Netherlands) are gratefully
acknowledged. This work was financially supported by a Séneca
Foundation grant to A.P. de los Ríos.
Notes and references
The ionic liquids were assayed for Cl− contents as follows: an
aqueous solution of potassium chromate (5 g L−1, 27 mM, indi-
cator) was added to the ionic liquid sample and a silver nitrite
solution (0.24 g L−1, 1.4 mM) was added dropwise. The end-
point was reached when a persistent red precipitate of silver
chromate was observed; the detection limit is approx. 100 nmol
Cl−. Less than 50 ppm of Cl− was present in the ionic liquids.
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precipitate had settled. The precipitate was washed twice with
acetone and dried over phosphorus pentoxide in vacuo overnight,
yield 1.8 mg, activity 38 U mg−1
.
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Acylation of 1-phenylethanol
In screw-capped vials of 2 mL total capacity, 1-phenylethanol
(12.0 μL, 0.1 mmol), vinyl acetate (18.4 μL, 0.2 mmol) and
1,2,3-trimethoxybenzene (8.6 mg, 0.05 mmol, internal standard)
were dissolved in the reaction medium (1 mL). Activated zeolite
KA (50 mg) and precipitated Novozym 525L (68 U) or CaLB
CLEA (6.4 mg, 68 U) were added. The mixture was shaken at
40 °C in an oil bath. At regular time intervals, 15 μL aliquots
were withdrawn and added to TBME (0.6 mL). The biphasic
mixture was vortexed during
1 min and centrifuged at
12 000 min−1. 5 μL samples of the supernatant were analyzed by
GC as described above. In all cases, a blank experiment was run
in the absence of enzyme to ensure that there was no background
reaction.
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