Continuous Flow Enzymatic Kinetic Resolution and Enantiomer Separation using Ionic Liquid/Supercritical Carbon Dioxide Media

Article abstract of DOI:10.1002/adsc.200303109

The combination of kinetic resolution in ionic liquids (IL) and selective extraction with supercritical carbon dioxide (scCO₂) provides a new approach for the separation of enantiomers as exemplified by the lipase-catalyzed esterification of chiral secondary alcohols. Excellent enantioselectivities are achieved upon conversion of alcohols 1a-e to the corresponding acetates 4a-e or laureates 5a-e using various modifications of the lipase from Candida antarctica (CaL-B) in imidazolium-based ionic liquids. The anion of the ionic liquid has a significant influence on the performance of the bio-catalyst with bis(trifluoromethanesulfonamide) [BTA] giving the best results. The acetates 4a-e can be extracted from the reaction mixture preferentially over the alcohols 1a-e with scCO₂ under certain conditions, but preparatively useful selectivities would require advanced multi-step extraction procedures. In contrast, efficient separation is possible with relatively simple equipment if alcohols 1a-e are extracted preferentially from their corresponding laureates 5a-e. A "green" continuous process for the resolution of racemic alcohols without the use of organic solvents was devised on the basis of these findings.

Full text of DOI:10.1002/adsc.200303109

FULL PAPERS
Continuous Flow Enzymatic Kinetic Resolution and Enantiomer
Separation using Ionic Liquid/Supercritical Carbon Dioxide
Media
a,
a
a, b
¡
Manfred T. Reetz, * Wolfgang Wiesenhˆfer, Giancarlo Francio, Walter
Leitnera, b,*
a
Max-Planck-Institut f¸r Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 M¸lheim/Ruhr, Germany
Fax: ( ‡ 49)-208-306-2985, e-mail: reetz@mpi-muelheim.mpg.de
b
Lehrstuhl f¸r Technische Chemie und Petrolchemie, Institut f¸r Technische und Makromolekulare Chemie, RWTH
Aachen, Worringer Weg 1, 52056 Aachen, Germany
Fax: ( ‡ 49)-241-802-1777, e-mail: leitner@itmc.rwth-aachen.de
Received: June 3, 2003; Accepted: August 28, 2003
Abstract: The combination of kinetic resolution in the reaction mixture preferentially over the alcohols
ionic liquids (IL) and selective extraction with super- 1a e with scCO2 under certain conditions, but
critical carbon dioxide (scCO2) provides a new preparatively useful selectivities would require ad-
approach for the separation of enantiomers as vanced multi-step extraction procedures. In contrast,
exemplified by the lipase-catalyzed esterification of efficient separation is possible with relatively simple
chiral secondary alcohols. Excellent enantioselectiv- equipment if alcohols 1a e are extracted preferen-
ities are achieved upon conversion of alcohols 1a e tially from their corresponding laureates 5a e. A
to the corresponding acetates 4a e or laureates 5a e ™green∫ continuous process for the resolution of
using various modifications of the lipase from Can- racemic alcohols without the use of organic solvents
dida antarctica (CaL-B) in imidazolium-based ionic was devised on the basis of these findings.
liquids. The anion of the ionic liquid has a significant
influence on the performance of the bio-catalyst with
bis(trifluoromethanesulfonamide) [BTA] giving the Keywords: ionic liquids; kinetic resolution; lipases;
best results. The acetates 4a e can be extracted from supercritical carbon dioxide; supercritical fluids
Introduction
the acylating agent.[6] In this case, the semi-ester formed
in the lipase-catalyzed reaction is soluble in the aqueous
Efficient methodologies for the production of enantio- phase, while the unreacted antipode remains in the
merically pure or enriched compounds are of great organic phase, allowing for convenient separation. In
academic and industrial importance, (bio)catalytic ki- yet another approach preferential partitioning of ester
netic resolution being an attractive option.[1,2] Lipase- and alcohol was achieved by using a highly fluorinated
catalyzed acylation of chiral alcohols is of particular acylating agent followed by separation in an organic/
interest as a preparative tool and as a benchmark fluorous biphasic system.[7] We introduce here a new
reaction.[1,3] The main disadvantage for practical appli- methodology based on the combination of an ionic
cation results from the necessity to use chromatography liquid (IL) and supercritical carbon dioxide (scCO2) as
for separation of the alcohol and ester, requiring the use the solvent system for reaction and separation.
and recycling or disposal of large volumes of organic
Several groups have demonstrated the feasibility of
solvents. Of course, if the particular system is amenable employing ionic liquids (ILs)[8] as solvents in enzymatic
to dynamic kinetic resolution, a separation problem catalysis,[9] including the use of lipases in the kinetic
does not arise.[4] If this is not possible, alternative resolution of chiral alcohols.[10] According to these
strategies need to be developed, and indeed, several reports, suspensions of lipases in ionic liquids can have
innovative suggestions have been made recently. For several advantages such as high enzyme stability and
example, scavenging of the unreacted alcohol on a solid sometimes even enhanced activity and stereoselectivity.
support was used to enable separation by subsequent However, although the actual kinetic resolution was
filtration.[5] In a different approach, batch-wise separa- performed in ionic liquids, organic solvents were in fact
tion by multistep liquid/liquid extraction was achieved used to remove the mixture of ester and non-reacted
in certain cases through the use of succinic anhydride as alcohol from the ionic liquid phase and further separa-
Adv. Synth. Catal. 2003, 345, 1221 1228
DOI: 10.1002/adsc.200303109
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Manfred T. Reetz et al.
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tion required conventional, chromatographic tech-
niques. Obviously, this defeats part of the original goal
as defined by the criteria of green chemistry.[11] Stimu-
lated by pioneering workof Beckman and Brennecke on
the phase behavior of IL/CO2,[12] we recently developed
a new and environmentally benign protocol for lipase-
catalyzed reactions in ionic liquids which makes use of
supercritical carbon dioxide (scCO2)[13] as a means to
extract the products in a completely solvent-free
form.[14,15] This approach allows the enzyme/ionic liquid
mixture to be recycled and re-used in batch-wise or
continuous-flow operations. Independently, Lozano
et al. described the combination of ionic liquids and
scCO2 in a lipase-catalyzed transesterification, whereby
the starting ester was injected into the system in the form
of a hexane solution.[16a] In other workthe influence of
compressed CO2 on the transesterification performance
of a-chymotrypsin in ionic liquids was studied by Laszlo
et al.[16b]
In the present paper, we describe in detail the
application of our new IL/scCO2 system for lipase-
catalyzed kinetic resolution of chiral secondary alcohols.
In particular, we report how this approach allows us to
combine (bio)catalytic esterification with highly effi-
cient enantiomer separation in a continuous flow
operation avoiding the use of organic solvents com-
pletely. The underlying principle of this approach is
outlined in Figure 1. The racemic alcohol and the
acylating agent are transported into the reactor using
scCO2 as the mobile phase. There, one of the enantiom-
ers is esterified selectively by the lipase in the ionic
liquid and the mixture of products is continuously
Figure 1. Strategies for enantiomer separation via (bio)cata-
lytic kinetic resolution and selective supercritical extraction
in an ionic liquid/scCO2 continuous flow system.
extracted with the scCO2 stream. Ester and unreacted non-coordinating anions are the best choice for enzy-
alcohol are then separated downstream by controlled matic catalysis.[9] Even within this class of ILs, the an-
density reduction via variation of temperature and/or ion has a significant influence on the performance of
pressure. Judicious choice of the acylating agent allows the bio-catalyst. With 1-butyl-3-methylimidazolium
modification of the molecular structure of the ester to [BMIM] as cation, the bis(trifluoromethanesulfon-
exhibit either higher or lower solubility in scCO2 than amide) anion [BTA] led to the highest selectivity with
the corresponding alcohol, which is crucial for efficient almost perfect discrimination between the two enan-
extractive separation. Vinyl laureate (3) was found to be tiomers. The selectivity was largely unaffected by the
a cheap acylation agent that renders the ester less choice of the enzyme modification. Surprisingly,[17] the
soluble than the alcohol, which allows for efficient activity was higher with the commercial lyophilized
separation.
biocatalyst as compared to the supported enzyme either
in form of Novozyme SP 435 or as a sol-gel immobili-
zate.[18] Nevertheless, the immobilized enzymes were
chosen for subsequent studies because they did not
result in significant transesterification beyond 50%
Results and Discussion
Our study focused on lipase B from Candida antarctica conversion even after prolonged reaction times.
(CaL-B) as a transesterification catalyst known to Initial experiments directed towards the selective
exhibit high activities and selectivities for a broad range separation of ester and unreacted alcohol with scCO2
of substrates in organic solvents.[1,3] Exploratory experi- were carried out for the kinetic resolution of 1-phenyl-
ments to select the optimum combination of ionic liquid ethanol (1b) with vinyl acetate (2) using the apparatus
and lipase modification were carried out using 2-octanol depicted in Figure 2. The system comprises a window-
(1a) and vinyl acetate (2) as standard substrates under equipped 10-mL stainless steel reactor that is heated,
conventional conditions (Scheme 1, Table 1). All ionic and agitation is ensured by a magnetic stirring bar.
liquids examined here resulted in high enzyme activity, Initially, the reactor is charged with the suspension of the
substantiating previous findings that ILs with lipophilic immobilized enzyme in the ionic liquid followed by the
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Enzymatic Kinetic Resolution and Enantiomer Separation
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Table 1. Influence of solvent and lipase-preparation on kinetic resolution of alcohol 1a.
Entry
Reaction Medium
Biocatalyst
ee (1a)[a]
[%]
ee (4a)[a]
[%]
Activity
Conversion [%]
[mmol minÀ1 gÀ1]
15 min
18 h
82.2
100
70.8
65.2
68 h
1
2
3
4
Isooctane
Lyophilized Cal-B
Lyophilized Cal-B
Lyophilized Cal-B
Lyophilized Cal-B
Lyophilized Cal-B
Novozyme SP 435
Sol-gel immobilizate
> 99.9
94.9
> 99.9
99.2
98.4
> 99.9
> 99.9
88.6
89.8
85.7
98.7
97.8
98.2
99.6
53.0
51.4
53.9
50.1
41.6
11.8
10.4
[BMIM][PF6]
[BMIM][BF4]
[BMIM][BTA]
[BMIM][BTA]
[BMIM][BTA]
[BMIM][BTA]
5[b]
6[b]
7[b]
5800
1190
1250
57.0
50.4
50.9
Quantities: 1a ˆ 0.5 mmol; 2 ˆ 1 mmol; biocatalyst ˆ 5 mg; reaction medium ˆ 1 mL. Reaction conditions: thermo mixer
1000 rpm; Tˆ 458C.
[a]
Measured at approximately 50% conversion.
Biocatalyst ˆ 2.5 mg.
[b]
Scheme 1. Stereoselective acylation of chiral alcohols.
respectively.[14] In agreement with the generally higher
CO2 solubility of acetates as compared to the corre-
sponding alcohols in scCO2,[19] we found that an enrich-
ment of ester (R)-4b can be achieved when extraction
from the IL is carried out at lower CO2 densities. A
maximum enrichment of (R)-4b:(S)-1b of 1.4:1 was
achieved in the CO2 stream at 608C and 90 bar in a single
extraction step. After extraction of about 65% of the
total ester formed, an alcohol rich fraction [(R)-4b:(S)-
1b ˆ 1:3] was obtained upon increasing the CO2 pres-
sure to 150 bar. Similarly, moderate levels of enrichment
could be achieved with 1-(2-naphthyl) ethanol (1e) as
Figure 2. Reaction/separation system for batch-wise combi-
nation of enzymatic kinetic resolution and enantiomer
separation using an ionic liquid/scCO2 medium.
substrate using this methodology [(R)-4e:(S)-1e ˆ 1.9:1
at 608C/110 bar; (R)-4e:(S)-1e ˆ 1:4.2 at 508C/185 bar].
In agreement with findings of other researchers, these
data suggest that a preparatively useful separation of
liquid substrate. After heating for the desired reaction alcohols and acetates obtained from enzymatic kinetic
time, CO2 extraction is carried out using a compressor resolutions requires the use of advanced multi-step
and a capillary tube dipping into the IL phase. After extraction procedures.[20] However, it appeared intrigu-
complete extraction, new substrate can be introduced by ing to us to improve the separation efficiency by
an HPLC pump without depressurization.
molecularly engineering the relative solubility of alcohol
As reported previously, batch-wise extraction with and ester in scCO2 (see Figure 1). The solubility of a
scCO2 at 408C and 110 bar allows the complete recovery compound in scCO2 depends critically on polarity, vapor
of a 1:1 mixture of (R)-4b and (S)-1b, each in high pressure, cohesive energy, and the presence of special
enantiomeric purity amounting to 95 and 99.6% ee, ™CO2-philic∫ groups.[21,22] In the present case, we decided
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Manfred T. Reetz et al.
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to concentrate on vapor pressure as the variable because
it is readily influenced by variation of the chain length of
the carboxylate group in the acylating agent. If the chain
is long enough, the low volatility of the molecule
dominates over the influence of polarity and the ester
should become less soluble in scCO2 than the corre-
sponding alcohol. Gratifyingly, a cheap and readily
accessible long-chain acylating agent is available in he
form of vinyl laureate (3) which is produced on a large
technical scale as a co-monomer for vinyl polymers.
Figure 3 demonstrates the viability of this concept for
the lipase-catalyzed kinetic resolution of 2-octanol (1a)
coupled with a simple isobaric extraction at 608C and
90 bar. In the early fractions, the alcohol (S)-1a is
extracted with high selectivity ((R)-5a:(S)-1a ˆ 1:18),
whereas the lauryl ester (R)-5a is obtained with high
purity in the later fractions [(R)-5a:(S)-1a ˆ 21:1).[23] In
order to speed up the extraction for the ester-rich
fraction, it is more convenient to increase the CO2
density after most of the alcohol has been isolated.
Figure 3. Separation of (S)-2-octanol [(S)-1a] and the lauryl
ester of the (R)-enantiomer [(R)-5a] obtained from lipase-
catalyzed kinetic resolution in [BMIM][BTA] by isobaric
extraction with scCO2 (608C/90 bar).
This simple and non-optimized procedure yields effi- vented through cryo-traps that were changed periodi-
cient levels of separation for a variety of secondary cally (total extraction time approximately 90 h). With
alcohols 1 and their corresponding lauryl esters 5 this simple procedure, 66% of the theoretical amount of
(Table 2). For example, in the case of 3-methyl-2- (S)-1b was isolated from the first four traps with an
butanol (1c) the enrichment reaches a ratio of (R)- enantiomeric purity > 99.9% and less than 0.5% con-
5c:(S)-1c ˆ 1:20 in the low density fractions (90 bar/ tamination with (R)-5b. The next two traps contained
608C) and (R)-5c:(S)-1c ˆ 14:1 in the high density small amounts of a mixed fraction, after which the
fractions (200 bar/458C). This corresponds to 95.2% pressure in the reaction and extraction chamber was
alcohol selectivity and 93.5% ester selectivity, each with increased stepwise to 200 bar, yielding a total of 89% of
high levels of enantiomeric purity of > 99.5% for (S)-1c the theoretical amount of (R)-5b (ee > 99.9%) with less
and 92.8% for (R)-5c, respectively (Table 2).
In order to increase the separation, an additional
than 1% contamination with alcohol (S)-1b.
Following the CO2 extraction, the mixture of ionic
separation chamber can be introduced between the liquid and biocatalyst remaining in the reactor can be re-
reactor and the cryo-trap of the apparatus depicted in used without apparent loss of activity and selectivity.[14]
Figure 2. This was applied to the kinetic resolution of This remarkable stability is independent of the alcohol
rac-1b. After transesterification with Novozyme SP 435 used. For rac-1c, nine consecutive batches were run with
in [BMIM][BTA] at 458C for seven hours, the resulting no significant changes in the enzyme×s performance, and
mixture of (S)-1b and (R)-5b was extracted at 608C and separation efficiencies of > 90% for alcohol and esters
105 bar into the separation chamber where the pressure were achieved in the selective extractions in each case. It
was reduced to 80 bar before the CO2 stream was finally was also possible to carry out several sequential trans-
Table 2. Results of kinetic resolution and extraction.
Substrate
Extraction Conditions
Selectivity for 1 [%]
Selectivity for 4 [%]
ee (1) [%]
ee (4) [%]
1a[a]
1b[b]
1c[c]
1d[d]
1e[e]
90 bar, 608C
94.8
93.5
95.2
95.4
75.8
95.5
66.3
93.5
72.5
69.2
> 99.5
98.9
> 99.5
65.0
85.2
> 99.5
92.8
88.2
75.0
90 bar, 608C/200 bar, 458C
75 bar, 608C/200 bar, 458C
100 bar, 608C/200 bar, 458C
110 bar, 608C/200 bar, 458C
98.2
Quantities: Novozyme SP 435 ˆ 100 mg; [BMIM][BTA] ˆ 3 mL; (rac)-1 ˆ 4 mmol; 3 ˆ 6 mmol. Reaction conditions:
[a]
65.5 h; Tˆ 608C; p ˆ 100 bar (CO2).
[b]
65.5 h; Tˆ ca. 208C; atmospheric pressure.
[c]
61 h; Tˆ ca. 208C; atmospheric pressure.
[d]
15 h; Tˆ ca. 208C; atmospheric pressure.
[e]
65.5 h; Tˆ 608C; p ˆ 110 bar (CO2).
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Enzymatic Kinetic Resolution and Enantiomer Separation
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esterifications with different alcohols using the same IL/ associated with another density reduction. The liquid
enzyme mixture. Encouraged by the long-term stability phase formed in the second chamber was recycled
of the biocatalytic system, we envisaged continuous- periodically into the first vessel. The chiral alcohol (S)-
flow operation of the combined kinetic resolution and 1b was separated continuously from the final CO2
separation procedure.[24]
stream upon venting through a cryo-trap, whereas the
The apparatus applied for continuous-flow processes ester (R)-5b accumulated in the first separation cham-
is depicted in Figure 4. A cascade of two reactors of ber.
different geometries was used in order to ensure
After continuous operation for 112 h, substrate deliv-
conversion close to the theoretical maximum of 50% ery was stopped and extraction continued until no more
without the necessity for detailed optimization of alcohol was contained in the CO2 stream. A total of 5.1 g
substrate and CO2 flow rates. The separation part (81% of theory) of (S)-1b was collected in the cryo-trap
consisted of two high pressure chambers connected with an ee of > 97% containing less than 0.1% of ester
with a recirculating pump and the possibility to intro- (R)-5b. From the first extractor, (R)-5b (15.2 g, 97% of
duce additional CO2 independently of the exit flow of theory) was obtained with an ee > 97% and <0.5% of
the reactor. Recycling of CO2 at the end of the process (S)-1b. After resuming the substrate flow, the process
was not implemented at this stage of the investigations, started again with identical activity and selectivity,
but is theoretically possible. The kinetic resolution of proving again the remarkable long-term stability of
racemic 1b was used as a model reaction to demonstrate the biocatalyst under the reaction and extraction con-
the principle features of the continuous reaction/sepa- ditions.
ration sequence.
The mixture of rac-1b and 3 was passed through the
reactor part in a stream of high density scCO2 as the Conclusions
mobile phase and transesterification occurred upon
contact with the biocatalyst suspended in the ionic liquid In summary, we have developed a new method for
[BMIM][PF6]. The CO2 stream was expanded into the enantiomer separation based on a combination of
first separation chamber where a liquid phase was (bio)catalytic kinetic resolution and supercritical fluid
formed due to the density reduction and concomitant extraction using an ionic liquid/scCO2 system. The
lowering of the solvent power of CO2. Extraction was interplay of molecular engineering and process optimi-
continued with additional CO2 to the second chamber zation makes this approach highly flexible as demon-
Figure 4. Reaction/separation system for continuous-flow combination of enzymatic kinetic resolution and enantiomer
separation using an ionic liquid/scCO2 medium.
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Manfred T. Reetz et al.
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changed periodically during the extraction procedure. Gen-
erally, the extraction conditions were initially set at high
temperature and low pressure (low density) and gradually
changed to higher densities by increasing the pressure and/or
lowering the temperature (see Table 2 and text for details).
Typically, CO2 flow rates were adjusted to 10 20 L ¥ hÀ1
(measured as exit flow after venting).
strated for the lipase-catalyzed esterification of racemic
secondary alcohols. Preparatively useful separation
efficiencies can be achieved in single step extractions
when vinyl laureate is used as cheap acylating agent
which reduces the CO2 solubility of the ester relative to
the alcohol. Excellent enantiomer separation, high
enantioselectivity and pronounced long-term stability
of the lipase are key features of the process. At the same
time, the methodology avoids the use of organic solvents
completely and allows for continuous flow operation.
We therefore believe that this novel approach provides a
promising basis for the development of environmentally
benign methodologies for the practical production of
enantiomerically pure or enriched compounds.
Continuous-Flow Combination of Kinetic Resolution
and Extraction with scCO2
In the apparatus shown in Figure 4, a mixture of rac-1b and 3
(molar ratio 1:2) was introduced into a stream of high density
scCO2 (508C/200 bar) using a HPLC pump at a flow rate of
0.6 mL ¥ hÀ1. The mobile phase passed through the cascade of
two small reactors (Vˆ 10 mL and 15 mL, respectively) that
were filled with immobilized CaL-B as a suspension in the ionic
liquid [BMIM][PF6] (3 mL and 8 mL, respectively). A mixture
of commercially available lipase immobilized Novozyme
SP 435 and a sol-gel immobilizate[18] was used in a 1:2 ratio.
Novozyme SP 435 floats on top of the ionic liquid, whereas the
sol-gel immobilizate is dispersed more homogeneously. The
total amount of immobilizate was 150 mg in the first and
600 mg in the second reactor.
Experimental Section
Caution
The handling of pressurized gases must be carried out only
using suitable equipment under appropriate safety conditions.
The CO2 stream was expanded into the first separation
chamber (Vˆ 225 mL, Tˆ 708C, p ˆ 130 bar) where a liquid
phase was formed. A constant exit flow was achieved by using a
gas dosing unit at the end of the reactor with a sampling volume
of 0.02 mL at a frequency of 0.1 sÀ1. Additional CO2 was
introduced after the dosing unit at the entrance of the first
separation chamber and extraction was continued into the
second chamber (Vˆ 100 mL, Tˆ 1008C, p ˆ 100 bar). The
liquid phase formed in the second chamber was recycled
periodically into the first vessel with an HPLC pump. After
leaving the second chamber, the final CO2 stream was vented
through a cryo-trap kept at 08C. The contents of the cryo-trap
and the first extraction chamber were collected for off line
analysis.
Materials and Analysis
The biocatalysts used in this study were obtained from Roche
(lyophilized CaL-B: Chirazyme L-2) and Novo Nordisk(im-
mobilized CaL-B: Novozyme SP 435) or prepared according to
known procedures (sol-gel immobilized CaL-B).[18] The ionic
liquids were purchased from Solvent Innovation, Germany,[25]
and used after filtration over a short path of silica using
methylene chloride as the mobile phase. The secondary
alcohols 1a e, vinyl acetate (2) and vinyl laureate (3) were
commercial products and used as received. Analysis of the
product mixtures for conversion and compositionwas achieved
1
by H NMR spectroscopy of CDCl3 solutions using a Bruker
DPX 300 or AMX 400 spectrometer. The enantiomeric purity
was determined by GC or HPLC in the Department of
Chromatography at the Max-Planck-Institut f¸r Kohlenfor-
schung.[26]
References and Notes
[1] a) H. G. Davies, R. H. Green, D. R. Kelly, S. M. Roberts,
Biotransformations in Preparative Organic Chemistry:
The Use of Isolated Enzymes and Whole Cell Systems in
Synthesis; Academic Press, London, 1989; b) C. H. Wong,
G. M. Whitesides, Enzymesin Synthetic Organic Chem-
istry, Vol. 12, Pergamon, Oxford, 1994; c) K. Drauz, H.
Waldmann (Eds.), Enzyme Catalysis in Organic Syn-
thesis: A Comprehensive Handbook, 3 Volumes, 2nd
edn., Wiley-VCH, Weinheim, 2002; d) K. Faber, Bio-
transformations in Organic Chemistry, 4th edn., Spring-
er, Berlin, 2000.
[2] Examples of non-enzymatic catalysis, see: a) E. Vedejs,
E. Rozners, J. Am. Chem. Soc. 2001, 123, 2428 2429;
b) B. Tao, J. C. Ruble, D. A. Hoic, G. C. Fu, J. Am. Chem.
Soc. 1999, 121, 5091 5092; c) E. R. Jarvo, G. T. Cope-
land, N. Papaioannou, P. J. Bonitatebus Jr., S. J. Miller, J.
Am. Chem. Soc. 1999, 121, 11638 11643.
Batchwise Kinetic Resolution and Subsequent
Extraction with scCO2
Experiments were carried out using the apparatus depicted in
Figure 2. A suspension of immobilized CaL-B (Novozyme
SP 435; 100 mg) in [BMIM][BTA] (3 mL) was added in a
window-equipped stainless-steel high pressure reactor (Vˆ
10 mL) equipped with a magnetic stirring bar, a thermocouple,
a pressure sensor and an inlet and outlet valve. The racemic
alcohol (4 mmol) and acylating agent (6 mmol) were then
introduced. The reaction mixture was stirred at the desired
temperature for a given time as indicated in Table 2.
After the desired conversion was reached, the products were
extracted by flushing CO2 via a capillary through the ionic
liquid. At the reactor exit, the CO2 stream was depressurized
using a heated needle valve and the organic materials were
collected from the gas stream in cryo-traps at 08C, which were
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Enzymatic Kinetic Resolution and Enantiomer Separation
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asc.wiley-vch.de
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1227

Manfred T. Reetz et al.
FULL PAPERS
[25] http://solvent-innovation.de.
thyl)ethyl acetate (4e), 25 m IVADEX-7/OV-1701.
HPLC: 1-phenylethyl laureate (5b) and 4-phenyl-2-butyl
laureate (5d), 250 mm; Chiralcel OD-H, 4.6 mm i. d.; 4-
phenyl-2-butanol (1d), 1-(2-naphthyl)ethanol (1e), and
1-(2-naphthyl)ethyl laureate (5e), 250 mm, Chiralcel
OD-R, 4 mm i.d.
[26] GC: 2-octanol (1a), 25 m LIPODEX A; 2-octyl laureate
(5a), 25 m PER ME-BCD/OV 225/ FS 611; 1-phenyl-
ethanol (1b), 1-phenylethyl acetate (4b), and 3-methyl-2-
butanol (1c), 25 m TBBCD/OV1701/ FS 610; 3-methyl-2-
butyl laureate (5c), 25 m IVADEX 1/PS086; 1-(2-naph-
1228
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2003, 345, 1221 1228