Application of ionic liquids in enzymic resolution by hydrolysis of cycloalkyl acetates

Article abstract of DOI:10.1016/j.tetasy.2006.10.045

A comparative study was performed in the enzymic resolution of the isomers of 2-(4-methoxybenzyl)cyclohexyl acetates 1 and 2. The investigation consisted in application of three commercially available lipases (Novozyme 435, Lipozyme IM and non-immobilized

Full text of DOI:10.1016/j.tetasy.2006.10.045

Tetrahedron: Asymmetry 17 (2006) 2987–2992
Application of ionic liquids in enzymic resolution
by hydrolysis of cycloalkyl acetates
a
b
b
b
ˇ
´
Epameinondas Xanthakis, Marie Zarevu´cka, David Saman, Martina Wimmerova,
Fragiskos N. Kolisisa and Zdeneˇk Wimmerb,*
aBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str.,
Zografou Campus, GR-15700 Athens, Greece
b
´
Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo na´meˇstı 2, CZ-16610 Prague 6, Czech Republic
Received 6 October 2006; accepted 30 October 2006
Available online 28 November 2006
Abstract—A comparative study was performed in the enzymic resolution of the isomers of 2-(4-methoxybenzyl)cyclohexyl acetates 1
and 2. The investigation consisted in application of three commercially available lipases (Novozyme 435, Lipozyme IM and non-immo-
bilized powdered lipase from Candida antarctica), two ionic liquids (1-butyl-4-methylpyridinium chloride and 1,3-dimethylimidazolinium
methyl sulfate), three modifications of the reaction conditions and two respective isomers of the racemic substrate (1 and 2), and resulted
in our finding the appropriate conditions to get both of the products, stereoisomers of 2-(4-methoxybenzyl)cyclohexanol (3; 1S,2S) or (5;
1S,2R), and (in some cases) also the stereoisomers of the deracemized substrate (4; 1R,2R) or (6; 1R,2S) with high or acceptable enan-
tiomeric purity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The objective of our study was to compare the lipase-med-
iated process for enzymic resolution of racemic substrates
under several modified conditions:
Biocatalytic processes, employing either enzymes or whole
cells for the biotransformation of many natural or syn-
thetic substrates, have been useful tools for the preparation
of important enantiopure synthons for decades.1 The
majority of organic compounds used as substrates in those
processes are insoluble in aqueous media, and, therefore,
useful procedures were elaborated for application of
enzyme technology in organic media, miscible or immisci-
ble with water.2 Organic solvents, which are not miscible
with water, often allow application of higher reaction
temperatures for enzymic processes than the enzyme would
allow in aqueous media.1,2 Most of the organic solvents
which may be used as media for enzymic processes are
not friendly for the environment. It seems that ionic liquids
represent certain environmentally friendly alternatives to
organic solvents. It was found that ionic liquids can be used
either as co-solvents in aqueous systems or in biphasic sys-
tems or even as pure solvents.3
(i) Performing the enzymic reaction in the presence of the
selected ionic liquids, 1-butyl-4-methylpyridinium
chloride ([BMPy]Cl) or 1,3-dimethylimidazolinium
methyl sulfate ([DMIM]MeSO4), as co-solvents in an
aqueous system.
(ii) Performing the experiments under the conditions
given in (i), with the additional presence of aceto-
nitrile, an organic solvent miscible with water.
(iii) Performing the experiments without the presence of
both ionic liquids and acetonitrile (the reference
experiments).
The separated cis- and trans-isomers of 2-(4-methoxy-
benzyl)cyclohexyl acetate (1 and 2) were employed as
substrates for the above-described modifications of the
lipase-mediated processes. The cis- and trans-isomers of
2-(4-methoxybenzyl)cyclohexanol are used as intermediates
in the synthesis of several series of biologically active com-
pounds displaying effect mostly on morphology or oviposi-
tion of insects.4 These biologically active compounds are
considered to represent environmentally friendly insect pest
*
Corresponding author. Tel.: +420 220183281; fax: +420 220183582;
e-mail: wimmer@uochb.cas.cz
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.10.045

2988
E. Xanthakis et al. / Tetrahedron: Asymmetry 17 (2006) 2987–2992
(a) Novozyme 435: This enzyme showed remarkable selec-
OCH3
OCH3
4
1
12
3
2
5
6
11
10
tivity in transformation of the cis-isomer of substrate 1.
Under the presence of [BMPy]Cl (entry 1), product 3
[(1S,2S); ee >99%] and the remaining substrate 4
[(1R,2R); ee >99%] were received in high yields and
with excellent enantiomeric purity. In turn, when
[DMIM]MeSO4 was used as a medium (entry 2) in
the same reaction, no progress was observed, and race-
mic starting substrate 1 remained untouched in the
reaction mixture. In the presence of acetonitrile as an
auxiliary solvent, the hydrolysis of 1 proceeded quanti-
tatively, giving almost racemic product 3 (entries 4 and
5). A reference experiment (entry 3), that is, the experi-
ment performed with the absence of both, the ionic
liquid and the auxiliary solvent, resulted in receiving
product 3 [(1S,2S); ee >99%] with high enantioselectiv-
ity but lower chemical yield, and due to that fact, the
remaining substrate 4 [(1R,2R); ee >62.5%] had a lower
enantiopurity. The same lipase mediated resolution of
the trans-isomer of substrate 2 with excellent enantio-
selectivity [5; (1S,2R); ee >99%] in the presence of both
ionic liquids (entries 6 and 7), and also in the reference
experiment (entry 8). A quantitative ester fission of 2
was observed. When the reaction was performed in
the presence of either ionic liquid tested in the presence
of acetonitrile as an auxiliary solvent, the product was
almost racemic (entries 9 and 10).
7
8
9
OCOCH3
OCOCH3
13 14
2
1
OCH3
OCH3
OCH3
OCH3
OH
OH
5
3
+
+
OCOCH3
OCOCH3
6
4
Scheme 1. Lipase-mediated resolution by hydrolytic reactions. Numbers
of carbon atoms in the compound 1 show carbon atom numbering used in
the assignment of the 1H and 13C NMR signals.
management agents.4 In this investigation, attention was
also focused on the preparation of enantiopure com-
pounds. Enzymic processes designed under different condi-
tions are often used for the preparation of convenient
enantiopure intermediates or final products with biological
activity. For that reason, isomers of 2-(4-methoxy-
benzyl)cyclohexanol are often used as convenient model
substrates in designing novel procedures and/or their
modifications in our projects.5
(b) Lipozyme IM: The effect of this enzyme on the derace-
mization reaction tested was remarkably different
from the effect of Novozyme 435. The reference exper-
iment (entry 13) was the only modification of the res-
olution reaction through which product 3 [(1S,2S);
ee >99%] was received with high enantioselectivity
and high chemical yield. This enzyme was blocked
by the presence of [BMPy]Cl (entry 11). The presence
of the alternative ionic liquid, [DMIM]MeSO4 (entry
12), resulted in poor chemical yield but high enantio-
meric purity of product 3 [(1S,2S); ee >99%]. The pres-
ence of acetonitrile together with either of the ionic
liquids resulted in no reaction progress (entries 14
and 15). A similar scheme was observed with substrate
2. No reaction was observed in the presence of
[BMPy]Cl (entry 16), and in the modification of the
reaction conditions using acetonitrile as an auxiliary
solvent (entries 19 and 20). However, using [DMIM]-
MeSO4 (entry 17) yielded more than double the quan-
tity of product 5 [(1S,2R); ee >99%] than the reference
experiment (entry 18).
In this investigation, a comparative study was performed
consisting of the application of three commercially avail-
able lipases [Novozyme 435 (lipase from Candida antarc-
tica, immobilized on acrylic resin), Lipozyme IM (lipase
from Mucor miehei, immobilized on a macroporous ion-ex-
change resin), and powdered, non-immobilized lipase from
C. antarctica], two ionic liquids ([BMPy]Cl and [DMIM]-
MeSO4), three modifications of the reaction conditions
(cf. above) and two respective isomers of the racemic sub-
strates 1 and 2 (Scheme 1).
(c) Non-immobilized lipase from C. antarctica: Using
substrate 1, this enzyme was blocked by [DMIM]-
MeSO4 (entry 22). No reaction was also observed in
the presence of acetonitrile (entries 24 and 25). The
presence of [BMPy]Cl (entry 21) resulted in substantial
ester 1 fission, however, with a poor enantioselectivity
[3, (1S,2S); ee = 19%]. A higher chemical yield of 3
[(1S,2S); ee >99%] and excellent enantiomeric purity
were found only in the reference experiment (entry
23). Using substrate 2, this enzyme was also blocked
by the presence of [DMIM]MeSO4 (entry 27), and
by the simultaneous presence of either of the ionic liq-
uids tested and acetonitrile (entries 29 and 30). Almost
quantitative ester 2 splitting, with poor enantioselec-
2. Results and discussion
The starting isomers of substrates 1 and 2 were prepared
from the respective racemic cis- and trans-isomers of 2-(4-
methoxybenzyl)cyclohexanol, using acetic anhydride and
pyridine, and subsequent chromatographic purification in
quantitative yields.
The key results of this investigation are summarized in
Table 1. The discussion of the results is presented accord-
ing to the lipases applied (entries mentioned in the para-
graphs below can be found in Table 1):

E. Xanthakis et al. / Tetrahedron: Asymmetry 17 (2006) 2987–2992
2989
Table 1. Results of the enzymic process
Entry Substrate Enzymea (specific activity; quantity
of the enzyme used for the reaction)
Ionic liquid/auxiliary organic solvent Products (yieldb [%]; eec); absolute configuration
1
2
3
4
5
1
1
1
1
1
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
[BMPy]Cl/none
[DMIM]MeSO4/none
None/none
[BMPy]Cl/acetonitrile
[DMIM]MeSO4/acetonitrile
3 (48; >99); (1S,2S); 4 (44; 92); (1R,2R)
3 (—; —); (1S,2S); 4 (96; —); (1R,2R)
3 (30; >99); (1S,2S); 4 (65; 62.5); (1R,2R)
3 (94; 0); (1S,2S); 4 (—; —); (1R,2R)
3 (95; 0); (1S,2S); 4 (—; —); (1R,2R)
6
7
8
9
10
2
2
2
2
2
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
Novozyme 435 (7 U mgꢀ1; 20 mg)
[BMPy]Cl/none
[DMIM]MeSO4/none
None/none
[BMPy]Cl/acetonitrile
[DMIM]MeSO4/acetonitrile
5 (45; >99); (1S,2R); 6 (48; 97.5); (1R,2S)
5 (43; >99); (1S,2R); 6 (49; 97); (1R,2S)
5 (49.5; >99); (1S,2R); 6 (49; >99); (1R,2S)
5 (93; 0); (1S,2R); 6 (—; —); (1R,2S)
5 (92; 0); (1S,2R); 6 (—; —); (1R,2S)
11
12
13
14
15
1
1
1
1
1
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [BMPy]Cl/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [DMIM]MeSO4/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) None/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [BMPy]Cl/acetonitrile
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [DMIM]MeSO4/acetonitrile
3 (—; —); (1S,2S); 4 (95; —); (1R,2R)
3 (17; >99); (1S,2S); 4 (72; 60); (1R,2R)
3 (45; >99); (1S,2S); 4 (49; 90.5); (1R,2R)
3 (—; —); (1S,2S); 4 (95; —); (1R,2R)
3 (—; —); (1S,2S); 4 (94; —); (1R,2R)
16
17
18
19
20
2
2
2
2
2
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [BMPy]Cl/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [DMIM]MeSO4/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) None/none
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [BMPy]Cl/acetonitrile
Lipozyme IM (0.03 U mgꢀ1; 20 mg) [DMIM]MeSO4/acetonitrile
5 (—; —); (1S,2R); 6 (96; —); (1R,2S)
5 (25; >99); (1S,2R); 6 (70; 55); (1R,2S)
5 (11; >99); (1S,2R); 6 (74.5; 31.5); (1R,2S)
5 (—; —); (1S,2R); 6 (96; —); (1R,2S)
5 (—; —); (1S,2R); 6 (97; —); (1R,2S)
21
22
23
24
25
1
1
1
1
1
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
[BMPy]Cl/none
[DMIM]MeSO4/none
None/none
[BMPy]Cl/acetonitrile
[DMIM]MeSO4/acetonitrile
3 (85; 19); (1S,2S); 4 (12; 20); (1R,2R)
3 (—; —); (1S,2S); 4 (94; —); (1R,2R)
3 (40; >99); (1S,2S); 4 (55; 75); (1R,2R)
3 (—; —); (1S,2S); 4 (96; —); (1R,2R)
3 (—; —); (1S,2S); 4 (93; —); (1R,2R)
26
27
28
29
30
2
2
2
2
2
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
CAL (3.1 U mgꢀ1; 4 mg)
[BMPy]Cl/none
[DMIM]MeSO4/none
None/none
[BMPy]Cl/acetonitrile
[DMIM]MeSO4/acetonitrile
5 (90; 6); (1S,2R); 6 (8; —); (1R,2S)
5 (—; —); (1S,2R); 6 (95; —); (1R,2S)
5 (25; 89); (1S,2R); 6 (69; 58.5); (1R,2S)
5 (—; —); (1S,2R); 6 (95; —); (1R,2S)
5 (—; —); (1S,2R); 6 (94; —); (1R,2S)
a CAL = non-immobilized lipase from C. antarctica.
b Hyphen in the column of yields means that the appropriate compound was not found during HPLC analysis.
c Hyphen in the column of enantiomeric excess (ee) means that this value could not be determined, that is, the appropriate product was either racemic or its
yield was too low to determine its ee value.
tivity, to the product 5 [(1S,2R); ee = 6%] was
observed in the presence of [BMPy]Cl (entry 26). Even
the reference experiment resulted in moderate enantio-
meric purity of product 5 [(1S,2R); ee = 89%].
came the results obtained from the appropriate reference
experiments (Table 1, entries 3 and 8).
Evaluating the rest of the experiments, only Lipozyme IM
in the presence of [DMIM]MeSO4 was able to produce 5
(Table 1, entry 17) in higher chemical yield than in the com-
parable reference experiment (Table 1, entry 18). Summa-
rizing the results from the rest of the experiments
presented in Table 1, the reference experiments (entries
13, 23 and 28) gave better results than any modification
of this process performed under the presence of the tested
ionic liquids. The presence of acetonitrile in the reaction
system contributed to the quantitative course of the enzy-
mic process (entries 4, 5, 9 and 10) mediated by Novozyme
435 in the presence of either ionic liquid. In turn, the pres-
ence of this organic co-solvent blocked the rest of the reac-
tions (entries 14, 15, 19, 20, 24, 25, 29 and 30), in which
either Lipozyme IM or non-immobilized lipase from C.
antarctica was used as a biocatalyst.
3. Conclusion
Novozyme 435 was able to mediate enzymic resolution by
hydrolysis of the racemic substrates 1 and 2 under the pres-
ence of [BMPy]Cl (Table 1, entries 1 and 6) affording either
products 3 [(1S,2S); ee >99%] and 4 [(1R,2R); ee = 92%] or
5 [(1S,2R); ee >99%] and 6 (1R,2S; ee = 97.5%). In addi-
tion, this reaction catalyzed by Novozyme 435 was success-
ful under the presence of [DMIM]MeSO4 only for
substrate 2 (Table 1, entry 7), affording products 5
[(1S,2R); ee >99%] and 6 [(1R,2S); ee = 97%]. The chemical
yields and the enantiomeric purity of products 3–6 result-
ing from these reactions (Table 1, entries 1, 6 and 7) over-

2990
E. Xanthakis et al. / Tetrahedron: Asymmetry 17 (2006) 2987–2992
In summary, it is possible to evaluate this complex study as
successful, because we found modifications of this enzymic
process by employing selected ionic liquids, through which
we obtained the requested products 3–6 enantiomerically
pure (ee >99%) and in high chemical yields.
to synthesize 1 lmol of propyl laurate per minute at
60 ꢁC. Non-immobilized lipase from C. antarctica (Fluka)
was used as the third enzyme tested, showing specific activ-
ity 3.1 U mgꢀ1; 1 U corresponds to the amount of enzyme
liberating 1 lmol of oleic acid per minute from triolein
used as a substrate at pH = 8.0 and at 40 ꢁC.
4. Experimental
4.1. General
4.3. 2-(4-Methoxybenzyl)cyclohexyl acetates 1 and 2
In a typical experiment, either of the respective isomers of
2-(4-methoxybenzyl)cyclohexanol (4.3 g; 19.52 mmol) was
dissolved in dry pyridine (20 mL), and acetic anhydride
(10 mL) was added. The reaction mixture was allowed to
stand overnight and then poured onto a mixture of ice
and hydrochloric acid (1:1; 30 mL). The organic layer
was extracted with toluene, dried over sodium sulfate and
the solvent was evaporated, leaving the crude residue.
Products 1 or 2 were received by a small column chroma-
tography in 95% yields [4.86 g (1) and 4.90 g (2), respec-
tively]. Their analytical data are summarized below.
1
The H NMR and the 13C NMR spectra were recorded
on a Bruker AVANCE 500 spectrometer (in FT mode)
at 500.1 and 125.8 MHz, respectively, in CDCl3 using
1
either tetramethylsilane (d 0.0 for H NMR) or a solvent
signal (CDCl3—d 77.00 for 13C NMR) as internal refer-
ence at a temperature 303 K. The 19F NMR spectra were
recorded on a Varian UNITY 500 spectrometer at
470.3 MHz in deuteriochloroform using hexafluoro-
benzene as external reference (d ꢀ162.9). 2D NMR exper-
iments were measured using the following characteristic
1
1
parameters: H,1H-PFG-COSY—spectral width 9 ppm in
Compound 1: H NMR (CDCl3): 1.19–1.27 (m, 1H, C-4),
both f1, f2 dimensions, delay 1 s, data matrix for process-
ing 2048 · 2048 data points; 1H,13C-PFG-HSQC—spec-
tral width 9 ppm in f2 and 180 ppm in f1, delay 1 s, data
matrix for processing 2048 · 2048 data points. IR spectra
were recorded in a solution (CCl4) on a Bruker IFS 88
instrument. Mass spectra (FAB) were recorded on a VG
analytical 70–250 SE mass spectrometer, ZAB–EQ
(BEQQ configuration) at 70 eV. Preparative column chro-
matography was performed on a silica gel type 60 (parti-
cle size 0.04–0.063 mm; Fluka, Switzerland). TLC was
performed on aluminium sheets precoated with silica gel
60 (Merck, Germany). Analytical HPLC was carried out
on a TSP (Thermoseparation Products, USA) instrument
equipped with a ConstaMetric 4100 Bio pump and a
SpectroMonitor 5000 UV DAD. The analyses of the
products were performed on a chiral Nucleodex b-OH
column (150 · 4 mm; Macherey-Nagel, Germany) using
a methanol/water mixture (9:1, v/v) as mobile phase at
0.3 mL minꢀ1. The eluate was monitored at 220, 254
and 275 nm, and the UV spectra were run from 200 to
300 nm. An Autopol IV polarimeter (Rudolph Research
Analytical, USA) was used for the measurement of optical
rotation. A Unimax 1010 incubator (Heidolph, Germany)
equipped with controlled heating was used to accommo-
date a magnetic stirrer under its cover to keep the re-
quested reaction temperature for proceeding of the
enzymic reactions.
1.35–1.44 (m, 1H, C-6), 1.40–1.53 (m, 2H, C-3), 1.45–1.54
(m, 2H, C-5), 1.65–1.75 (m, 1H, C-4), 1.70 (m, 1H, C-2),
1.88–1.94 (m, 1H, C-6), 2.10 (s, 3H, C-14) 2.40 (dd,
J = 8.0, 13.7, 1H, C-7), 2.56 (dd, J = 6.9, 13.7, 1H, C-7),
3.78 (s, 3H, C-12), 4.90 (dt, J = 2.0, 2.0, 4.6, 1H, C-1),
6.81 (m, 2H, C-10), 7.01 (m, 2H, C-9); 13C NMR (CDCl3):
20.89 (t, C-5), 21.30 (q, C-14), 24.96 (t, C-4), 26.95 (t, C-3),
29.89 (t, C-6), 37.64 (t, C-7), 42.42 (d, C-2), 55.21 (q, C-12),
72.21 (d, C-1), 113.73 (d, C-10), 129.87 (d, C-9), 132.49 (s,
C-8), 157.84 (s, C-11), 170.66 (s, C-13); IR (CCl4): 3064 (w),
3032 (w), 2936 (s), 1737 (s), 1613 (m), 1513 (s), 1246 (s),
1041 (s), 948 (w); FAB MS: (m/z) 262 ([M]+, 17), 203
(24), 121 (100), 91 (5). For C16H22O3 (262.34) calcd: C,
73.25; H, 8.46. Found: C, 73.29; H, 8.44.
1
Compound 2: H NMR (CDCl3): 0.97 (ddt, J = 3.5, 12.3,
12.3, 14.0, 1H, C-3), 1.06–1.15 (m, 1H, C-5) 1.24–1.35
(m, 2H, C-4 and C-6), 1.57–1.63 (m, 1H, C-4), 1.66–1.74
(m, 3H, C-2, C-3 and C-5), 1.99–2.02 (m, 1H, C-6), 2.02
(s, 3H, C-14), 2.22 (dd, J = 9.0, 13.7, 1H, C-7), 2.83 (dd,
J = 4.0, 13.7, 1H, C-7), 3.78 (s, 3H, C-12), 4.56 (dt,
J = 4.7, 10.0, 10.0, 1H, C-1), 6.82 (m, 2H, C-10), 7.03 (m,
2H, C-9); 13C NMR (CDCl3): 21.30 (q, C-14), 24.53 (t,
C-5), 25.10 (t, C-4), 30.01 (t, C-3), 31.86 (t, C-6), 37.95 (t,
C-7), 43.83 (d, C-2), 55.23 (q, C-12), 76.89 (d, C-1),
113.61 (d, C-10), 130.04 (d, C-9), 132.29 (s, C-8), 157.79
(s, C-11), 170.82 (s, C-13); IR (CCl4): 3064 (w), 3033 (w),
2936 (s), 1735 (s), 1613 (m), 1513 (s), 1244 (s), 1028 (s),
876 (w), 854 (w); FAB MS: (m/z) 262 ([M]+, 13), 203 (15)
202 (16), 121 (100), 91 (6). For C16H22O3 (262.34) calcd:
C, 73.25; H, 8.46. Found: C, 73.22; H, 8.49.
4.2. Enzymes
Three enzymes were used in this study. Lipozyme IM
(Novo Nordisk, Denmark) is the lipase from Rhizomucor
miehei immobilized by adsorption on a macroporous ion
4.4. Enzymic resolution of 2-(4-methoxybenzyl)cyclohexyl
acetates 1 and 2
exchange resin, showing specific activity 0.03 U mgꢀ1
;
1 U corresponds to the amount of enzyme liberating
1 lmol of oleic acid per minute from triolein used as a sub-
strate at pH = 8.0 and at 40 ꢁC. Novozyme 435 (Novo
Nordisk, Denmark) is the lipase from C. antarctica immo-
bilized on a polyacrylate resin, showing specific activity
7 U mgꢀ1; 1 U corresponds to the amount of enzyme able
In a typical reference experiment (Table 1, entries 3, 8, 13,
18, 23 and 28), the racemic substrate (1 or 2; 20 mg;
0.09 mmol) was mixed with phosphate buffer (1.7 mL;
pH = 6.5), and the enzyme was added (for its quantity
see Table 1). Either an ionic liquid (382 mg; 0.3 mL) was

E. Xanthakis et al. / Tetrahedron: Asymmetry 17 (2006) 2987–2992
2991
added, where needed (Table 1, entries 1, 2, 4–7, 9–12,
14–17, 19–22, 24–27, 29 and 30), or even acetonitrile
(0.7 mL), the auxiliary solvent, was added (Table 1, entries
4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29 and 30). Complete mis-
cibility of either aqueous phase with ionic liquid, or aque-
ous phase, ionic liquid and acetonitrile was observed. The
reaction mixture was stirred for 72 h at 40 ꢁC. Work-up
of the reaction mixture consisted of filtering the enzyme
off the reaction mixture. The rest of the mixture was
extracted with ether, dried over sodium sulfate, then the
solvent was evaporated and the residue was either sepa-
rated by column chromatography (Table 1, entries 1, 3,
6–8, 13, 17, 23 and 28) to give pure products (3–6; Scheme
1) and/or analyzed by chiral HPLC. The chemical yields of
products 3–6, as well as their enantiomeric purity and abso-
lute configuration are given in Table 1. The analytical data
of 3 and 5 are summarized below.
4.5. Chemical hydrolysis of 2-(4-methoxybenzyl)cyclohexyl
acetate (4 and 6) to 2-(4-methoxybenzyl)cyclohexanols 7 and
8
For analytical purpose, samples of the deracemized
substrates 4 and 6 were chemically hydrolyzed to the corre-
sponding stereoisomers of 2-(4-methoxybenzyl)cyclohexa-
nol. A solution of the respective compounds 4 or 6
(10 mg; 0.018 mmol) in a mixture of methanol (1 mL)
and water (0.25 mL) was heated at reflux in the presence
OCH3
OCH3
OCOCH3
OCOCH3
6
4
1
Compound 3: H NMR (CDCl3): 1.18–1.25 (m, 1H, C-4),
1.34–1.44 (m, 2H, C-3), 1.42–1.48 (m, 2H, C-5 and C-6),
1.54–1.62 (m, 1H, C-5), 1.64–1.70 (m, 2H, C-2 and C-4),
1.73–1.79 (m, 1H, C-6), 2.48 (dd, J = 7.6, 13.6, 1H, C-7),
2.66 (dd, J = 7.6, 13.6, 1H, C-7), 3.78 (s, w = 11, 1H, C-
1), 3.79 (s, 3H, C-12), 6.82 (m, 2H, C-10), 7.10 (m, 2H,
C-9); 13C NMR (CDCl3): 20.34 (t, C-5), 25.31 (t, C-4),
26.37 (t, C-3), 33.27 (t, C-6), 37.75 (t, C-7), 43.67 (d, C-
2), 55.23 (q, C-12), 68.51 (d, C-1), 113.66 (d, C-10),
129.95 (d, C-9), 133.01 (s, C-8), 157.75 (s, C-11); IR
(CCl4): 3631 (w), 3501 (w), 3064 (w), 3032 (w), 2934 (s),
2835 (m), 1176 (s), 1041 (s), 974 (m); FAB MS: (m/z)
OCH3
OCH3
OH
OH
8
7
OCH3
OCH3
220 ([M]+). For C14H20O2 (220.30) calcd: C, 76.32; H,
20
9.15. Found: C, 76.29; H, 9.12. ½aꢁD ¼ þ34:4 (c 0.116,
O-MTPA
O-MTPA
CHCl3).
10
9
1
Compound 5: H NMR (CDCl3): 0.90 (ddt, J = 3.5, 11.5,
OCH3
OCH3
11.5, 16.5, 1H, C-3), 1.09 (dtt, J = 3.5, 3.5, 11.7, 11.7,
15.0, 1H, H-5), 1.19–1.29 (m, 2H, C-4 and C-6) 1.46 (m,
1H, C-2), 1.56–1.60 (m, 1H, H-5), 1.64 (ddt, J = 2.0, 3.5,
3.5, 16.5, 1H, C-3), 1.68–1.73 (m, 1H, C-4), 1.95–2.00 (m,
1H, C-6), 2.33 (dd, J = 9.0, 13.5, 1H, C-7), 3.07 (dd,
J = 4.1, 13.5, 1H, C-7), 3.28 (dt, J = 4.3, 9.9, 9.9, 1H, C-
1), 3.79 (s, 3H, C-12), 6.82 (m, 2H, C-10), 7.10 (m, 2H,
C-9); 13C NMR (CDCl3): 24.88 (t, C-4), 25.43 (t, C-5),
30.00 (t, C-3), 35.77 (t, C-6), 38.07 (d, C-7), 47.08 (t, C-
2), 55.22 (q, C-12), 74.51 (d, C-1), 113.61 (d, C-10),
130.25 (d, C-9), 132.67 (s, C-8), 157.76 (s, C-11); IR
(CCl4): 3624 (w), 3604 (w), 3064 (w), 3033 (w), 2835 (m),
1177 (m), 1042 (s), 1025 (m); FAB MS: (m/z) 220 ([M]+).
5
OH
OH
3
OCH3
OCH3
O-MTPA
O-MTPA
11
12
Scheme 2. Reactions performed in the synthesis of the (R)-MTPA ester of
chiral alcohols for analytical purposes.
For C14H20O2 (220.30) calcd: C, 76.32; H, 9.15. Found:
C, 76.35; H, 9.11. ½aꢁD ¼ ꢀ21:3 (c 0.083, CHCl3).
20
Table 2. Selected 1H and 19F NMR parameters (signals and coupling constants) of 9–12
Ester of the (R)-MTPA
Parameter
Product (absolute
configuration)
1H NMR
19F NMR
d (H-7a)
d (H-7b)
J (2,7a)
J (2,7b)
d (CF3)
9
10
11
12
2.34
2.18
2.25
2.08
2.51
2.89
2.45
2.70
8.2
9.9
8.1
9.8
6.7
3.2
6.8
3.1
ꢀ67.31
ꢀ67.55
ꢀ67.13
ꢀ67.44
7 (1R,2R)
8 (1R,2S)
3 (1S,2S)
5 (1S,2R)

2992
E. Xanthakis et al. / Tetrahedron: Asymmetry 17 (2006) 2987–2992
of potassium carbonate (15 mg) for 2 h. Methanol and
water were removed under reduced pressure, the residue
was applied onto a silica gel column and purified, affording
the pure products 7 and 8 in the yields P95%.
the Greek General Secretariat of Research and Technol-
ogy. The authors thank Novo Nordisk (Denmark) for a
generous gift of Lipozyme IM and Novozyme 435.
4.6. Synthesis of 3,3,3-trifluoro-2-methoxy-2-phenylpropa-
noic acid esters of 2-(4-methoxybenzyl)cyclohexanols 9–12
References
1. Klibanov, A. M. Nature 2001, 409, 241–246.
2. (a) Ballesteros, A.; Bornscheuer, U.; Capewell, A.; Combes,
D.; Condoret, J.-F.; Koening, K.; Kolisis, F. N.; Marty, A.;
Menge, U.; Scheper, T.; Xenakis, A. Biocatalysis 1995, 13,
1–42; (b) Cabral, J. M. S.; Aires-Barros, M. R.; Pinheiro, H.;
Prazeres, D. M. F. J. Biotechnol. 1997, 59, 133–143; (c) Carrea,
G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39, 2226–2254.
3. Kragl, U.; Eckstein, M.; Kraftzik, N. Biocatalytic Reactions in
Ionic Liquids. In Ionic Liquids in Synthesis; Wasserscheid, P.,
Welton, T., Eds.; Wiley-VCH: Weinheim, 2003; pp 336–347.
A general procedure used for the synthesis of the (R)-
MTPA (3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acid;
Mosher’s acid) esters on a milligram scale starting from the
(S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride
(MTPCl, Mosher’s chloride) was carried out as described
previously.5,6 Esters 9–12 (Scheme 2) were obtained in
1
quantitative yields and their H and 19F NMR data used
for the assignment of the absolute configuration of the par-
ent major enantiomers 3, 5, 7 and 8 of 2-(4-methoxy-
benzyl)cyclohexanol are given in Table 2 and discussed in
Section 2.
ˇ
´
´
´
4. (a) Wimmer, Z.; Saman, D.; Kuldova, J.; Hrdy, I.; Bennettova,
B. Bioorg. Med. Chem. 2002, 10, 1305–1312; (b) Wimmer, Z.;
´
´
´
Kuldova, J.; Hrdy, I.; Bennettova, B. Insect Biochem. Mol.
Biol. 2006, 36, 442–453.
5. Wimmer, Z.; Skouridou, V.; Zarevu´cka, M.; Saman, D.;
ˇ
Kolisis, F. N. Tetrahedron: Asymmetry 2004, 15, 3911–3917,
and the references cited therein.
Acknowledgements
6. (a) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969,
34, 2543–2549; (b) Dale, J. A.; Mosher, H. S. J. Org. Chem.
1970, 35, 4002–4003; (c) Dale, J. A.; Mosher, H. S. J. Am.
Chem. Soc. 1973, 95, 512–519; (d) Sullivan, G. R.; Dale, J. A.;
Mosher, H. S. J. Org. Chem. 1973, 38, 2143–2147.
This work was supported by the Czech-Greek project
KONTAKT (2003–2005). Its Czech part (Project ME
692) was funded by the Czech Ministry of Education,
and its Greek part (Project EPAN 4.3.6.1) was funded by