One-pot enzymatic resolution and separation of sec-alcohols based on ionic acylating agents

Article abstract of DOI:10.1002/anie.200701774

(Figure Presented) Going their different ways: A simple, robust, efficient, and reusable system for the one-pot preparative resolution and separation of sec-alcohols is described. The system is based on the combination of sequential enzymatic kinetic resolution (lipase B from Candida Antarctica, CALB) and transesterification in ionic liquids (ILs) with an ionic acylating agent 1 and removal of each enantiomer by extraction with an organic solvent.

Full text of DOI:10.1002/anie.200701774

Communications
DOI: 10.1002/anie.200701774
Biocatalysis
One-Pot Enzymatic Resolution and Separation of sec-Alcohols Based
on Ionic Acylating Agents**
Nuno M. T. Lourenço and Carlos A. M. Afonso*
Enantiomerically pure alcohols are important intermediates
in the pharmaceutical industry for the production of active
pharmaceutical ingredients. On a large scale, and asides from
chiral natural resources, they can be obtained by classical
chemical asymmetric or biotechnological processes. The
approaches used most often are the asymmetric hydrogena-
tion of ketones,[1] Corey–Bakshi–Shibata borane reduction,[2]
the kinetic resolution of epoxides,[3] enzymatic resolution,[4]
ketone reduction,[5] and fermentation.[6]
holding a lower fluorous phase that serves a barrier to
separate two organic phases was used. One side contained the
source phase MeOH/CH3Cl, and the other side contained a
receiving phase containing MeOH/MeOÀ. A mixture of S-
fluorous ester (99% ee) and the unreacted R-alcohol (91%
ee) obtained from EKR was added to the source side of the U-
tube, and it was possible to separate each enantiomer of
alcohol with only a slight loss in ee (about 2–4%). In 2006,
Matsumoto and co-workers described an EKR approach
based on monomethoxy-poly(ethylene glycol)-supported car-
bonates as anchoring agents.[13] Such a suitable water-soluble
supported carbonate containing one of the enantiomers could
be separated from the R-alcohol by hexane extraction. By
repeating the extraction with EtOAc, it was then possible to
remove from the water layer the supported carbonate
containing one of the enantiomers.
During the course of our recent investigations, Salunkhe
and co-workers have reported another similar EKR involving
anchored ionic liquids containing a carboxylate group in
DMSO.[14] Ibuprofen-anchored ionic liquids were resolved,
and (S)-ibuprofen was isolated in 87% yield (86% ee).
Over the last few years, the use of ionic liquids (ILs) to
replace organic solvents for EKR has gained much attention
and appears to be a good alternative.[15–22] The high stability of
ionic liquid media, the ease of product recovery combined
with enzyme stability,[23] and the possibility to reuse the
reaction media make ILs a powerful tool for biocatalysis.
Additionally, task-specific ionic liquids (TSILs) have shown a
considerable advantage in several reactions.[24]
Herein, we report a practical, reusable, and efficient
process for the resolution and separation of both enantiomers
of sec-alcohols, without the need for laborious chromatog-
raphy separation, based on the combination of TSILs as
acylating and anchoring ionic liquids and an enzyme working
sequentially as a kinetic resolution agent and to detach one
enantiomer of the alcohol at the end (Scheme 1).
A central feature of this EKR and separation of alcohols
is the combination of an ionic liquid and an ionic acylating
agent. An acylating agent was envisaged that contained two
distinct parts, namely, an ionic moiety and an ester moiety
recognized by an enzyme, to allow the selective resolution
and separation of an ionically anchored ester from the
unreacted alcohol. The major advantage of this process
compared with other procedures described before is the
possibility to separate both free enantiomers of a racemic
mixture only by enzymatic resolution in a one-pot reaction
using one equivalent of acylating agent. This approach takes
advantage of the unique properties of ILs, that they are
formed only by ions, which provides an ionic pool crucial for
entrapping one of the enantiomers as an ionic ester moiety.
Enzymatic kinetic resolution (EKR) of racemic alcohols is
a well-established method and often the unique and/or most
practical route for the preparation of enantiomeric alcohols,
especially when both enantiomers are needed.[7,8] However, in
this method, some limitations need to be circumvented to
improve economical and environmental aspects. One prob-
lem is generally the need of a large excess of acylating agent
to achieve the desired conversions, with consequent gener-
ation of reactive side products that can change the reaction
course. Another major drawback in this transformation is the
product separation step. The enantiomers, one as an alcohol
and the other as an ester, formed during the acylation are
usually separated by chromatographic techniques. On the
laboratory scale this is not a problem, but on a large scale this
approach presents serious limitations.[9,10] To circumvent this
disadvantage, several new approaches have been reported;
for example, Theil and co-workers described the efficient
enzymatic resolution of sec-alcohols using fluorous acylating
agents.[11,12] To isolate the products, namely the R-fluorous
ester (98% ee) and the unreacted S-alcohol (99% ee), from
the reaction, repeated extractions with a fluorous solvent
were needed. In 2001, Curran, Theil, and co-workers
described another attractive kinetic resolution process involv-
ing fluorous triphasic reactions[12] whereby a simple U-tube
[*] Prof. C. A. M. Afonso
Centro de Química-Física Molecular, Complexo 1
Departamento de Engenharia Química e Biológica
Instituto Superior TØcnico
1049-001 Lisboa (Portugal)
Fax: (+351)21-841-7122
E-mail: carlosafonso@ist.utl.pt
N. M. T. Lourenço
REQUIMTE, Departamento de Química
Faculdade de CiÞncias e Tecnologia
Universidade Nova de Lisboa
2829-516 Caparica (Portugal)
[**] We thank the Fundaç¼o para a CiÞncia e Tecnologia (POCI 2010)
and FEDER (SFRH/BD/18487/2004; POCTI/QUE/35437/1999,
POCI/QUI/57735/2004) for financial support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or fromthe author.
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Chemie
R enantiomers in good yields and with good
enantiomeric excesses (Table 1). The best
result was generally obtained by combina-
tion of the acylating agent 4b and the ionic
liquid [bmim][PF6] containing the same
PF6À anion. Better yields and enantioselec-
tivities were obtained when longer reaction
times were applied for the first enzymatic
transformation (96 h). (S)-1-Phenylethanol
was isolated in 51% yield (80.9% ee), and
its R enantiomer was isolated in 41.3%
yield (99.3% ee). The first enzymatic trans-
esterification (step 1) turned out to be
slower than the second (step 2).
The resolution can also be performed
using the acylating agent 4a without sol-
vent, as 4a is liquid at room temperature.
However, in this case, it was observed that
the viscosity of the reaction mixture
increases with reaction time, forming a
slurry after 48 h and providing both enan-
tiomers in moderate yield and ee (step 1:
Scheme 1. Methodology for the enzymatic resolution and separation of sec-alcohols.
CALB=lipase B from Candida antarctica.
Additionally, as a result of ILs being immiscible with several
organic solvents, the unreacted alcohol can be removed by
repeated extraction with a common organic solvent. The fact
that ILs are almost non-volatile[25] allows the ethanol formed
during transesterification to be evaporated, thus moving the
equilibrium toward the formation of products. It is also known
that ILs provide a stable and friendly environment for
enzymes, which thereby retain their catalytic activity. The
anchored enantiomer that results as the product of step 1
(Scheme 1) can be removed in step 2 by a second enzymatic
transesterification (reversible reaction) using a primary
alcohol, such as EtOH.
Scheme 2. Synthesis of an acylating agent based on the imidazolium
cation. 1) EtOH, H2SO4, toluene, reflux, Dean–Stark apparatus, 15 h,
90%; 2) methyl imidazole, iPr2O, 708C, 48 h, 98%; 3) NaBF4 (4a),
KPF6 (4b), CH2Cl2, RT, 48 h, 4a 87%; 4b 74%.
After screening several poten-
Table 1: Enzymatic resolution and separation of racemic sec-alcohols using CALB as a biocatalyst.[a]
tial acylating agent candidates con-
taining different cations and alkyl
chains, the imidazolium cation
appeared the most appropriate
for this transformation. The acylat-
ing agent 4 can be obtained simply
by nucleophilic substitution of
ethyl 11-bromoundeconate 2 (see
Scheme 2). The effect of anion
type in the acylating agent and
solvent was disclosed by using
enzymatic resolution of 1-phenyl-
ethanol with CALB as a model
transformation, which allowed the
identification of 4a and 4b, and the
ionic liquids [bmim][PF6] and
[bmim][BF4] (bmim = 1-n-butyl-3-
methylimidazolium) as the best
candidates (Table 1).
Racemic
alcohol
Acylating
agent
IL[b]
Step 1: S enantiomer
Step 2: R enantiomer
t [h]
yield [%]
ee [%][c]
t [h]
yield [%]
ee [%][c]
R=Ph
4a
[bmim][PF6]
48
67.0
68.8
51.0
74.3
64.5
50.5
51.6
51.5
54.5
62.2
80.9
56.2
39.4
62.4
60.0
38.5[e]
24
24
24
24
24
24
24
24
25.9
22.5
41.3
19.2
19.4
33.2
30.6
25.0
88.9
98.5
99.3
98.9
99.0
96.4
91.9
96.5[e]
96[d]
96[d]
96[d]
48
4b
4a
4a
4a
4b
4b
[bmim[PF6]
[bmim][BF4]
–
[bmim][PF6]
[bmim][PF6]
[bmim][PF6]
R=Ph(CH2)2
R=n-C8H17
96
96
96
The use of different combina-
tions of acylating agent 4a/4b with
[a] All reactions were carried out in IL (0.8 mL) with alcohol (0.41 mmol), acylating agent (0.41 mmol),
and CALB (20 mg) at 358C. [b] Initial water content: 1.9 mg H2O per mL [bmim][PF6]; 3.8 mg H2O per mL
[bmim][BF4]. [c] Determined by HPLC. [d] Result corresponds to the first cycle of reuse of the reaction
medium (enzyme, acylating agent, IL). [e] Determined by GC of the corresponding butyrate ester.
[bmim][PF6]/[bmim][BF4]
and
CALB allowed us to obtain S and
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Table 2: Reuse of the reaction medium for the enzymatic resolution and separation of 1-phenylethanol (yields and ee values are given as
percentages).[a]
Acylating IL[b]
agent
Step 1: (S)-1-phenylethanol[c]
Step 2: (R)-1-phenylethanol[c]
1st cycle
yield ee
2nd cycle
3rd cycle
4th cycle
yield ee
1st cycle
yield ee
2nd cycle
3rd cycle
4th cycle
yield ee
yield ee
yield ee
yield ee
yield
ee
4b
4a
4a
[bmim][PF6] 51.0 80.9 56.9 75.2 62.3 71.1 73.2
51.0 41.3 99.3 43.1 99.2 44.9 98.7 38.4
97.9
[bmim][PF6] 68.8 62.2 58.3 63.0 62.5 57.2 63.3[d] 64.3 22.5 98.5 36.4 97.0 40.0 99.2 42.5[e] 97.6
[bmim][BF4] 74.3 56.2 64.5 58.7 76.7 50.4 64.2[f] 58.3 19.2 98.9 38.8 92.6 35.6 98.6 34.2[g] 85.3
[a] All reactions were carried out in IL (0.8 mL) with 1-phenylethanol (0.41 mmol), acylating agent (4a, 4b; 0.41 mmol), and CALB (20 mg) at 358C;
step 1: 96 h, 100 mmHg, S enantiomer removed by extraction with Et2O; step 2: EtOH (2.5 equiv), 24 h, R enantiomer removed by extraction with
Et2O. [b] Initial water content: 1.9 mg H2O per mL [bmim][PF6]; 3.8 mg H2O per mL [bmim][BF4]. [c] Enantiomeric excess (ee) determined by HPLC.
[d] 5th cycle: 62.9% yield, 59.6% ee; 6th cycle: 79.7% yield, 45.0% ee. [e] 5th cycle: 39.3% yield, 99.1% ee; 6th cycle: 32% yield, 85.2% ee. [f] 5th cycle:
76.1% yield, 43.1% ee. [g] 5th cycle: 23.5% yield, 76.8% ee.
64.5% yield, 39.4% ee; step 2: 19.4% yield, 99.0% ee).
Comparing these results with the ones in the presence of the
IL demonstrates that the IL solvent plays an important role in
that a better homogenization of the reaction medium is
achieved.
This methodology was applied to different substrates.
When 4-phenyl-2-butanol was used as substrate, both free
enantiomers were obtained: the S enantiomer (step 1: 51.6%
yield, 60% ee) and the R enantiomer (step 2: 30.6% yield,
91.9% ee). 2-n-Decanol provided (S)-2-decanol in 51.5%
yield and 38.5% ee (step 1) and (R)-2-decanol in 25% yield
and 96.5 ee (step 2). These results demonstrate the excellent
ability of this enzymatic resolution in the separation of
different substrates.
choosing an appropriate combination of enzyme and ionic
anchoring agent.
Experimental Section
General procedure for enzymatic kinetic resolution and separation of
sec-alcohols: CALB (Novozym 435; 20 mg) and racemic sec-alcohol
(0.41 mmol) were added to a stirred solution of acylating agent (4a–b;
0.41 mmol) in ionic liquid (0.8 mL). The reaction mixture was stirred
for 4 days under reduced pressure (100 mmHg) at 358C in a
thermostatic bath. After this time, the reaction mixture was extracted
with Et2O (3 ” 7 mL) and the organic phases were collected and
passed through a pipette-sized column packed with silica gel. The
solvent was then evaporated under reduced pressure to give the S-
configured sec-alcohol.
The reaction mixture was dried under reduced pressure
(20 mmHg) for 2 h. After this time, EtOH (2.5 equiv) was added
and the mixture was stirred for 1 day at 358C in a thermostatic bath.
Then, the reaction mixture was extracted again with Et2O (3 ” 7 mL),
and the organic phase was collected and passed through a pipette-
sized column packed with silica gel. The solvent was then evaporated
under reduced pressure to give the R-configured sec-alcohol.
Reuse experiment: The reaction mixture was dried under
reduced pressure (20 mmHg) for 2 h, then more racemic sec-alcohol
(0.41 mmol) was added to the mixture, and the reaction was
performed as described above.
The possibility of reusing the reaction medium (the
enzyme, the ionic acylating agent, and the ionic liquid) was
studied for the best observed acylating agents, 4a and 4b, and
ionic liquids, [bmim][PF6] and [bmim][BF4], using 1-phenyl-
ethanol as a model substrate (Table 2). Under these condi-
tions, it was possible to perform up to four (4b/[bmim][PF6]),
five (4a/[bmim][BF4]), and six (4a/[bmim][PF6]) consecutive
enzymatic resolution–separation steps using the same cata-
lytic reaction medium, without considerable decrease in the
resolution efficiency, which corresponds respectively to eight,
ten, and twelve overall enzymatic reactions. Further optimi-
zation of each cycle with respect to a specific substrate such as
an enzyme, the reaction time, and the temperature should
allow each enantiomer to be obtained in high yield and with
high enantiomeric excess.
In conclusion, we have demonstrated a new, simple,
reliable, reusable, and efficient preparative methodology for
the one-pot resolution–separation of secondary alcohols
based on the enzymatic resolution and temporal attachment
of one enantiomer to an ionic acylating reagent in an ionic
liquid environment, both of which are non-volatile thus
allowing non-reversible transformation by removal of ethanol
under vacuum. This attachment allows a selective extraction
of the free enantiomer by solvent extraction owing to the
ionic liquid environment. Subsequently, the attached enan-
tiomer is released by a second enzymatic transesterification
with ethanol, which regenerates the reaction mixture of active
enzyme, ionic acylating agent, and ionic liquid for another
reaction cycle. This method could be adapted to other target
substrates and extended to other functional groups by
Received: April 22, 2007
Published online: September 21, 2007
Keywords: alcohols · enzymes · ionic liquids · kinetic resolution ·
.
separation processes
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