Enzymic resolution of 2-substituted cyclohexanols through lipase-mediated esterification

Article abstract of DOI:10.1016/j.tetasy.2004.11.009

Several lipases were used for the kinetic resolution of the racemic cis- and trans-isomers of 2-(4-methoxybenzyl)cyclohexanol, by lipase-mediated esterification of the substrates to the corresponding acetate isomers. Lipase from Rhizomucor miehei was foun

Full text of DOI:10.1016/j.tetasy.2004.11.009

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3911–3917
Enzymic resolution of 2-substituted cyclohexanols through
lipase-mediated esterification
a,
b
a
a
ˇ
*
ˇ
Zdenek Wimmer, Vasso Skouridou, Marie Zarevu´cka, David Saman and
Fragiskos N. Kolisisb
a
´
ˇ ´
Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo namestı 2, CZ-16610 Prague 6, Czech Republic
bBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Street,
Zografou Campus, GR-15700 Athens, Greece
Received 24 September 2004; accepted 4 November 2004
Abstract—Several lipases were used for the kinetic resolution of the racemic cis- and trans-isomers of 2-(4-methoxybenzyl)cyclohex-
anol, by lipase-mediated esterification of the substrates to the corresponding acetate isomers. Conversion of the products and the
remaining deracemized substrates into diastereoisomeric esters of 3,3,3-trifluoromethyl-2-methoxy-2-phenylpropanoic acid, their
1
analysis by chiral HPLC and assignment of their absolute configurations through their H and 19F NMR spectra, were the basis
of evaluation of the studied enzymic process. Lipase from Rhizomucor miehei (RML) was found to be the most efficient enzyme
regarding enantiomeric excess (ee) and yield of the desired products, while resolution by lipase from Rhizopus arrhizus (RAL)
resulted in satisfactory ee and lower yields.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
methoxybenzyl)cyclohexanone mediated by different
microorganisms, mostly yeasts.16–22 The opposite stereo-
isomeric pair of the alcohols, however, is accessible
through laborious processes, and, generally, in lower
chemical yields and enantiomeric excesses, even if suc-
cessful resolutions have already been performed as
well.6,8–11,13,14
Biocatalysis in non-aqueous media has received consider-
able attention over the past decade,1,2 especially for the
synthesis of enantiopure compounds of biological inter-
est by lipases (triacylglycerol hydrolases, EC 3.1.1.3).3,4
In particular, the enzymic resolution by lipases of chiral
2-substituted cycloalkanols, precursors of the insect
juvenile hormone bioanalogues, has been studied exten-
sively.5–14 Among the lipases tested to date, lipase B
from Candida antarctica was the only one able to resolve
both separated racemic isomers of 2-(4-meth-
oxybenzyl)cyclohexanol15 with almost quantitative
enantiomeric excess (ee >99%) of the products, that is,
2-(4-methoxybenzyl)cyclohexyl acetates. This enzyme is
known to be a highly enantioselective catalyst for the
resolution of secondary alcohols, and a study15 has pro-
ven its ability in connection with the studied substrates.
Therefore, the objective of the present study was to find
lipases and reaction conditions, through which the re-
quired stereoisomers, (1R,2R)- and (1R,2S)-2-(4-meth-
oxybenzyl)cyclohexanol, would be accessible with high
enantiomeric excesses and chemical yields, preferably
among remaining substrates in the enzymic transesterifi-
cation. Starting from the separated racemic cis- and
trans-isomers of 2-(4-methoxybenzyl)cyclohexanol, suit-
able lipases are expected to catalyze the esterification of
the (1R,2R)- and (1R,2S)-2-(4-methoxybenzyl)cyclohex-
anol isomers to the corresponding acetates, while
(1S,2S)- and (1S,2R)-2-(4-methoxybenzyl)cyclohexanol
isomers will remain intact in the reaction mixture. The
basic goal of this study was to answer the question, if
the enzymic resolution can be managed in a way to
afford both the products and the remaining substrates,
with as high as possible enantiomeric excess and chemi-
cal yields.
In fact, two of the stereoisomers [(1S,2S )- and (1S,2R)-
2-(4-methoxybenzyl)cyclohexanol] of the possible are
easily accessible through asymmetric reduction of 2-(4-
*
Corresponding author. Tel.: +42 0220 183 281; fax: +42 0224 310
090; e-mail: wimmer@uochb.cas.cz
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.009

3912
Z. Wimmer et al. / Tetrahedron: Asymmetry 15 (2004) 3911–3917
2. Results and discussion
2-methoxy-2-phenylpropanoic acid (MTPA, Mosher
acid).23–26 The diastereoisomeric mixtures of the MTPA
esters 8a and 8b and 10a and 10b–12a and 12b (Scheme
2) can be separated by analytical HPLC and the content
of each enantiomer of the chiral alcohol present in the
samples calculated, based on the respective peak areas
of the diastereoisomeric MTPA esters in the HPLC
chromatograms. The absolute configurations of the
major enantiomers of the MTPA esters 8a and 10a–
12a can be assigned by measuring their 1H and 19F
NMR spectra.27 Enantiomeric purity and the absolute
configuration of the major enantiomers of all products
and deracemized substrates (Schemes 1 and 2) were
determined by applying this approach. Assignment of
the absolute configuration at the C(2) stereogenic centre
was based on the differences of the chemical shifts of the
signals of both hydrogen atoms of the CH2–Ar (benzyl)
group, which are not equivalent due to their chiral envi-
ronment (Fig. 1). This corresponds with the literature
data,23 and it has been proven several times during
our studies12–18 that an upfield shift of the signals of
Several lipases were used for the resolution of racemic
cis- and trans-2-(4-methoxybenzyl)cyclohexanol. Lip-
ases from Candida cylindracea (CCL), Penicillium roque-
fortii (PRL) and Rhizopus niveus (RNL) were unable to
catalyze the desired transesterification. The rest of the
lipases tested were able to resolve the racemates; Table
1 summarizes the results of the enzymic reactions, which
resulted in obtaining certain quantities of the products
and deracemized substrates. These reactions were sub-
jected to a more detailed study, during which the reac-
tion course was monitored using chiral HPLC, with
the products analyzed after final work-up of the reaction
mixtures in order to assign the absolute configuration of
both the major enantiomers of the products and the
deracemized substrates.
Determination of the enantiomeric purity of the major
enantiomers 3a and 4a of the products 3ab and 4ab
(Scheme 1) was accomplished in two ways. The first
method consisted of a synthesis of convenient diastereo-
isomeric derivatives of the alcohols 5a and 5b, 6a and 6b,
7a and 7b and 9a and 9b (Scheme 2). The second method
consisted of the application of a chiral stationary phase
of a cyclodextrin type to separate enantiomers of the
chiral alcohols (cf. Schemes 1 and 2) using chiral HPLC
analysis. A combination of both methods is convenient
to have as it gives two independent analytical methods
for assigning the absolute configuration of the major
enantiomers of chiral alcohols.
1
benzyl hydrogen atoms in the H NMR spectra of the
diastereoisomeric esters derived from (R)-MTPA, with
the esters derived from (S)-MTPA consistent only with
the (S)-absolute configuration at the C(1) carbon centre
of 2-(4-methoxybenzyl)cyclohexanol, as in 5a, 6a, 7b
and 9b.12–18,23 The evaluation of the 19F NMR data of
the diastereoisomeric esters resulted in the same conclu-
sion (Fig. 1). A downfield shift of the signal of the CF3
group in the (R)-MTPA esters in comparison with the
same shift observed in the (S)-MTPA esters is in agree-
ment with the expected displacement of the CF3 group
from the eclipsed arrangement with the carbonyl group
as a consequence of steric interactions of the bulkier
groups, that is, from the phenyl group of the MTPA
Convenient diastereoisomeric derivatives are the dia-
stereoisomeric esters of the chiral alcohols with the
respective pure enantiomers of (R)-(+)-3,3,3-trifluoro-
OCH3
OCH3
OH
O-MTPA
5a
11a
OCH3
OCH3
HO
O
F3C
OH
O-MTPA
O-MTPA
Ph
5b
11b
12a
OCH3
(R)-MTPA
OCH3
OCH3
OH
6a
OCH3
OCH3
OH
O-MTPA
6b
12b

Z. Wimmer et al. / Tetrahedron: Asymmetry 15 (2004) 3911–3917
3913
Table 1. Results of the enzyme-mediated resolutions applied to the respective racemic isomers 1 and 2 of 2-(4-methoxybenzyl)cyclohexanol
Entry
Substrate
Enzyme (appl. activity; weight)
Solvent
Products (yield [%];
enantiomeric excess [%]);
absolute configuration
1
2
3
4
5
1
2
1
2
1
RML (600mU; 20mg)
RML (600mU; 20mg)
RML (600mU; 20mg)
RML (600mU; 20mg)
RAL (40mU; 20mg)
Hexane
Hexane
Toluene
Toluene
Hexane
3a (36.5; 92.2); (1R,2R)
5a (48.0; 51.2); (1S,2S)
4a (38.3; 85.3); (1R,2S)
3a (5.8; >99.0); (1R,2R)
4a (8.0; 95.1); (1R,2S)
3a (18.3; 94.6); (1R,2R)
6a (50.1; 62.4); (1S,2R)
5a (49.7; 5.52); (1S,2S)
6a (50.7; 10.2); (1S,2R)
5a (50.6; 23.9); (1S,2S)
6
2
1
2
1
2
1
2
1
2
1
2
RAL (40mU; 20mg)
MJL (40mU; 8mg)
MJL (40mU; 8mg)
WGL (4U; 40mg)
WGL (4U; 40mg)
PPL (561.2U; 40mg)
PPL (561.2U; 40mg)
CLL (40mU; 40mg)
CLL (40mU; 40mg)
CLL (75mU; 75mg)
CLL (75mU; 75mg)
Hexane
Hexane
Hexane
4a (17.7; 73.5); (1R,2S)
3a (4.6; 95.0); (1R,2R)
4a (6.7; 93.6); (1R,2S)
No reaction
6a (54.0; 31.2); (1S,2R)
5a (50.9; 6.7); (1S,2S)
6a (51.0; 9.3); (1S,2R)
7
8
9
Hexane
Hexane
10
11
12
13
14
15
16
4a (1.8; n.d.a); —
6a (50.9; 3.6); (1S,2R)
5a (51.9; 8.8); (1S,2S)
6a (53.2; 14.5); (1S,2R)
Hexane
Hexane
3a (4.6; 93.0); (1R,2R)
4a (8.1; 90.4); (1R,2S)
Hexane
Hexane
No solventa
No solventa
No reaction
No reaction
5a (50.3; 0.8); (1S,2S)
3a (0.2; n.d.b); —
4a (1.8; n.d.b); —
6a (50.8; 3.5); (1S,2R)
a Vinyl acetate was used both, as the solvent and the acyl donor.
b n.d. = not determined.
OCH3
OCH3
+
OCH3
OAc
OH
3a
5a
OCH3
OCH3
OCH3
OCH3
+
+
OH
1
OAc
OAc
OH
OH
3b
4a
5b
6a
OCH3
OCH3
OCH3
+
OH
2
OAc
OH
4b
6b
Scheme 1.
part of the esters, and from the benzyl substituent at
C(2) of the alcoholic part of the MTPA esters. Selected
data are given in Table 2.
from the chiral HPLC analysis were in good agreement
with those obtained by analyzing the diastereoisomeric
MTPA esters of the studied enantiomers of the alcohols.
The latter method, based on the chromatographic
behaviour of the enantiomeric mixtures of the alcohols,
in which the major enantiomers correspond to the struc-
tures 5a, 6a, 7a and 9a using a chiral HPLC Nucleodex
b-OH column, filled with a chiral b-cyclodextrin-based
stationary phase, was applied to confirm the above-
described structure assignment. The results obtained
Detailed analysis of the samples derived from all the
reaction systems, as described above, showed that the
most successful enzyme in mediating the desired resolu-
tion was lipase from Rhizomucor miehei (RML) and two
reaction systems were used: (a) in the first system, hex-
ane was used as solvent and vinyl acetate as the acyl
donor (Table 1, entries 1 and 2). RML gave the best

3914
Z. Wimmer et al. / Tetrahedron: Asymmetry 15 (2004) 3911–3917
OCH3
OCH3
OCH3
OAc
OH
O-MTPA
3a
7a
8a
OCH3
OCH3
OCH3
OAc
OAc
OH
OH
O-MTPA
O-MTPA
3b
4a
7b
9a
8b
OCH3
OCH3
OCH3
10a
10b
OCH3
OCH3
OCH3
OAc
OH
O-MTPA
4b
9b
Scheme 2.
Figure 1. Models of the MTPA esters 11a–12b. More shielded H–CH0Ar, H0–CHAr hydrogen atoms and C–F3 atoms 11a and 12a; less shielded H–
CH0Ar, H0–CHAr hydrogen atoms and C–F3 atoms 11b and 12b.
results using both substrates. The maximum conversion
was achieved after 140h and the reactions were stopped
after 213h (for substrate 1) or 288h (for substrate 2).
The absolute configuration of the products was assigned
using NMR analysis of the diastereoisomeric MTPA
esters. Products 3a and 3b and 4a and 4b, were subjected

Z. Wimmer et al. / Tetrahedron: Asymmetry 15 (2004) 3911–3917
3915
Table 2. The 1H and 19F NMR based assignment of the absolute
configuration of the (R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoic
acid (MTPA) esters
vinyl acetate as acyl donor, no products were detected
(Table 1, entries 13 and 14). Therefore, the reaction sys-
tem was altered and vinyl acetate used to act as both
solvent and acyl donor (Table 1, entries 15 and 16),
and the amount of the enzyme increased to about dou-
ble quantity. In that case, small quantities of both
products were obtained as indicated in Table 1. Finally,
when lipases from C. cylindracea (CCL), Rhizopus niveus
(RNL) and P. roquefortii (PRL) were employed as reac-
tion mediators, no reaction or low yields of the products
were observed. RNL was used in two reaction systems
(hexane/vinyl acetate and vinyl acetate/vinyl acetate sys-
tem). No detailed analysis of the reaction products was
performed.
Compound d [H–CH0Ar]a d [H0–CHAr]a d (CF3)b ACc
5a
7a
6a
9a
2.25
2.33
2.07
2.17
2.45
2.52
2.70
2.89
ꢁ67.13
ꢁ67.31
ꢁ67.44
ꢁ67.55
1S,2S
1R,2R
1S,2R
1R,2S
a 1H NMR data based on the chemical shifts of the signals of the
hydrogen atoms in the benzyl (CH2Ar) group of the alcohol part of
the MTPA esters.
b 19F NMR data based on the chemical shifts of the signals of the
fluorine atoms in the CF3 functionality of the acid part of the MTPA
esters.
c AC = absolute configuration of the alcohols 5a, 6a, 7a and 9a, the
major enantiomers of 2-(4-methoxybenzyl)cyclohexanol.
3. Conclusion
to alkaline removal of the acyl functionality, and to sub-
sequent esterification of the resulting chiral alcohols 7a
and 7b and 9a and 9b with (S)-3,3,3-trifluoro-2-meth-
oxy-2-phenylpropanoyl chloride affording the (R)-
MTPA esters 8a and 8b and 10a and 10b–12a and 12b
(Scheme 2). These (R)-MTPA esters were analyzed
Two of the enzymes tested, lipases from R. miehei
(RML) and R. arrhizus (RAL), gave the only positive
results in mediating of the requested reaction. Both
enzymes accommodated the substrates in the hexane
or toluene (solvents)/vinyl acetate (acyl donor) system.
RML proved the most successful enzyme and the
achieved enantiomeric purity values (ee 92% for 3a,
and 85% for 4a) at 36% or 38% yields, respectively,
are encouraging results for obtaining the enantiomers
with the requested absolute configuration; (1R,2R) for
3a and (1R,2S) for 4a. In fact, when changing hexane
as the solvent for toluene, the enantiomeric excesses of
the products 3a and 4a increased to even more successful
values, exceeding ee >95% for both, 3a and 4a, however
the conversion of the enzymic process decreased to 6–8%
yield of 3a and 4a. Lipase RAL also showed its ability to
mediate this enzymic resolution with quite good results
regarding the enantiomeric purity of products 3a and
4a, however, their chemical yields were about a half of
those achieved with RML in hexane. All other enzymes
tested in this study showed results of lower importance
due to low or no conversions found.
1
through their H and 19F NMR spectra as described
above. The absolute configurations of the products were
assigned, and are given in Table 1. Chiral HPLC analy-
sis of the MTPA esters resulted in the calculation of the
enantiomeric purity of the parent products and derace-
mized substrates, which are summarized in Table 1;
(b) in the second reaction system, toluene was used as
the solvent and vinyl acetate as the acyl donor (Table
1, entries 3 and 4). The results indicate that toluene is
relatively inconvenient solvent for the enzyme, since
only 6–8% of the products were obtained. The data on
the absolute configurations and the ee values are shown
in Table 1.
The next successful enzyme was lipase from Rhizopus
arrhizus (RAL). Using hexane as solvent and vinyl
acetate as the acyl donor, this enzyme was able to medi-
ate the requested chiral resolution of substrates 1 and 2
(Table 1, entries 5 and 6). Maximum conversion was
achieved after 140h and the products obtained being
3a and 3b (18.3%) and 4a and 4b (17.7%). The conver-
sion of the enzymic reaction mediated by lipase from
Mucor javanicus (MJL) was lower than 10%, using hex-
ane as solvent, and vinyl acetate as acyl donor (Table 1,
entries 7 and 8). The yields of the products isolated from
this reaction were 4.6% (3a and 3b) and 6.7% (4a and
4b). The data on the absolute configurations and the
ee values are shown in Table 1. Low yields of both prod-
ucts were achieved in the processes of resolution medi-
ated by lipase from porcine pancreas (PPL): 4.6% (3a
and 3b) and 8.1% (4a and 4b), respectively (Table 1,
entries 11 and 12). In this enzymic process, hexane was
used as solvent and vinyl acetate as the acyl donor.
Lipase from wheat germ (WGL) did not seem able to
accommodate substrate 1, since there was no evidence
of reaction using TLC analysis (Table 1, entry 9). More-
over, only traces of the products 4a and 4b were found
when substrate 2 was used in the hexane/vinyl acetate
system (Table 1, entry 10). When lipase from Candida
lipolytica (CLL) was used, with hexane as solvent and
The (1S,2S)- and (1S,2R)-enantiomers of 2-(4-meth-
oxybenzyl)cyclohexanol are accessible with accept-
able enantiomeric excess (ee >95%) through enzymic
reduction of 2-(4-methoxybenzyl)cyclohexanone with
yeasts.16–22 There have been several lipases tested so far
(those from Rhizopus oryzae, C. cylindracea, porcine
pancreas or Geotrichum candidum), which enable us to
prepare the additional existing stereoisomers of 2-(4-
methoxybenzyl)cyclohexanol, that is, (1R,2R)- and
(1R,2S)-enantiomers with acceptable enantiomeric ex-
cess (ee >95%), but lower conversion (ꢀ30%).6,8–11,13,14
Herein we have extended the number of lipases, which
can be employed in the processes of resolution of 2-sub-
stituted cyclohexanols. Due to the changes in the strains
of the yeasts available for enzymic reductions, and due to
the modifications in commercial availability of isolated
lipases, accompanied by the existence of lipases prepared
in laboratories (not available commercially), we are re-
quired to periodically perform the most recent studies
of enzymic reductions and enzymic resolutions to inves-
tigate the potential of novel microorganisms and lipases
for substrates, which had been studied in the past.

3916
Z. Wimmer et al. / Tetrahedron: Asymmetry 15 (2004) 3911–3917
4. Experimental
and the residue applied onto a silica gel column and
purified, to afford the pure products 7a and 7b and 9a
and 9b in the yields P90%.
4.1. General
Small vials (up to 5mL inner volume) were used for per-
forming the enzymic reactions. The vials were equipped
with screw stopcocks and septa covered by a Teflon
4.5. Synthesis of 3,3,3-trifluoro-2-methoxy-2-phenylprop-
anoic acid esters of 2-(4-methoxybenzyl)cyclohexanol
1
layer. The H NMR and the 19F NMR spectra were
A general procedure used for the synthesis of the (R)-
MTPA (3,3,3-trifluoro-2-methoxy-2-phenylpropanoic
acid; MosherÕs acid) esters on a milligram scale starting
from the (S)-3,3,3-trifluoro-2-methoxy-2-phenylpropa-
noyl chloride (MTPCl, MosherÕs chloride) was carried
out as previously described.23–26 The esters 8a and 8b
and 10a and 10b–12a and 12b (Scheme 2) were obtained
recorded on a Varian UNITY 500 spectrometer (in a FT
mode) at 499.8 and 470.3MHz, respectively, in deuterio-
chloroform using either tetramethylsilane (d 0.0) as the
internal reference or hexafluorobenzene as external ref-
erence (d ꢁ162.9). Preparative column chromatography
was performed on a silica gel type 60 (particle size 0.04–
0.063mm; 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. Analysis of the chiral
products was performed on a chiral Nucleodex b-OH
column (150 · 4mm; Macherey-Nagel, Germany) using
methanol/water (4:1, v/v) as the mobile phase at
0.3mLminꢁ1. The eluate was monitored at 220, 254
and 275nm while the UV spectra were run from 200
to 300nm.
1
in quantitative yields and their H and 19F NMR data
used for the assignment of the absolute configuration
of the parent major enantiomers 5a and 5b, 6a and 6b,
7a and 7b and 9a and 9b of 2-(4-methoxybenzyl)cyclo-
hexanol, given in Table 2 and discussed in Section 2.
Acknowledgements
This work was supported by the Czech-Greek project
KONTAKT. Its Czech part (project ME 692) was
funded by the Czech Ministry of Education and its
Greek part (project EPAN 4.3.6.1) funded by the Greek
General Secretariat of Research and Technology.
4.2. Lipases
Nine lipases were used for the kinetic resolution of
the racemic cis- and trans-2-substituted cyclohexanols:
lipase from C. cylindracea (CCL; 1.6Umgꢁ1), C. lipo-
lytica (CLL; 0.001Umgꢁ1), M. javanicus (MJL;
0.005Umgꢁ1), P. roquefortii (PRL; 0.002Umgꢁ1), Rhi-
zomucor miehei (Lipozyme IM; RML; 0.03Umgꢁ1), R.
arrhizus (RAL; 0.002Umgꢁ1), Rhizopus niveus (RNL;
0.0025Umgꢁ1), lipase from porcine pancreas (type II;
PPL; 14.03Umgꢁ1) and from wheat germ (WGL;
0.1Umgꢁ1). Lipase RML (adsorbed on a macroporous
resin) was kindly offered by Novo Nordisk (Baegsvaerd,
Denmark) and lipase PPL obtained from Sigma (Stein-
heim, Germany). All other lipases were used in their free
forms and were generous gifts from Fluka Chemie
GmbH (Buchs, Switzerland).
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