A novel lipase enzyme panel exhibiting superior activity and selectivity over lipase B from Candida antarctica for the kinetic resolution of secondary alcohols

Article abstract of DOI:10.1016/j.tetasy.2012.03.013

A novel, commercially available lipase enzyme panel performing kinetic bioresolutions of a number of secondary alcohols is reported. The secondary alcohols that have been chosen are known from the literature to be particularly challenging substrates to resolve. Following initial screening, four co-solvents were investigated for each lead enzyme in an effort to assess their tolerance to common organic solvents. The superiority of these novel enzymes over lipase B from Candida antarctica (CALB) has been demonstrated.

Full text of DOI:10.1016/j.tetasy.2012.03.013

Tetrahedron: Asymmetry 23 (2012) 583–586
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A novel lipase enzyme panel exhibiting superior activity and selectivity
over lipase B from Candida antarctica for the kinetic resolution of secondary
alcohols
Maeve O’Neill a, Denis Beecher a, David Mangan a, , Andrew S. Rowan a, Agnieszka Monte b, Stefan Sroka b,
⇑
Jan Modregger b, Bhupinder Hundle b, Thomas S. Moody a
a Almac, Biocatalysis Group, David Keir Building, Stranmillis Road, Belfast, Northern Ireland, BT9 5AG, UK
b EUCODIS Bioscience GmbH, Campus Vienna Biocenter II, Viehmarktgasse 2 a/2. OG, 1030 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel, commercially available lipase enzyme panel performing kinetic bioresolutions of a number of
secondary alcohols is reported. The secondary alcohols that have been chosen are known from the liter-
ature to be particularly challenging substrates to resolve. Following initial screening, four co-solvents
were investigated for each lead enzyme in an effort to assess their tolerance to common organic solvents.
The superiority of these novel enzymes over lipase B from Candida antarctica (CALB) has been
demonstrated.
Received 14 March 2012
Accepted 27 March 2012
Available online 27 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
OR
OR
OR
1. Introduction
Although the growth of carbonyl reductase technology1–3 offers
increasing access to chiral alcohols, often in quantitative yield and
in enantiopure form, there are cases in which the formation of the
racemic alcohol can be achieved in a far more facile manner than
the synthesis of the analogous prochiral ketone. In this instance,
racemate resolution can prove to be more economically viable than
asymmetric reduction. This fact, coupled with the value of hydro-
lase enzymes as a tool to perform bio-polish ee enhancement when
required, should ensure their longevity for many years to come.
The use of hydrolase enzyme mediated esterification in the res-
olution of chiral secondary alcohols has been well documented in
the literature over the past few decades and has found widespread
use both in industry and academia.4–6 The vast majority of litera-
ture examples report lipase B from Candida antarctica (CALB) as
being the most suitable lipase enzyme for this purpose.7–13 Never-
theless, there are a number of substrates against which CALB dis-
plays either very poor activity, or selectivity, or both.14,15 It was
our intention to test a novel lipase panel containing 23 enzymes
against substrates 1a–5a (Fig. 1) that are known to be particularly
difficult to resolve with the hope of achieving their successful
kinetic resolution. Reported data on the analogous CALB mediated
reactions are shown in Table 1.
1
2
3
OR
OR
1a-5a: R=H
1b-5b: R=Ac
O
Cl
4
5
Figure 1. Secondary alcohols 1a–5a that are known to be difficult to kinetically
resolve, and their corresponding acetyl esters 1b–5b.
catered for by the industry benchmark, CALB. For example, the CALB
mediated kinetic resolution of compounds 1a–3a generally requires
high enzyme loading, long reaction times, and achieves only moder-
ate E values (with compound 2a being the exception exhibiting a
reasonably good E value albeit with an extremely slow reaction
rate). On the other hand, the CALB mediated acylation of compound
4a proceeds rapidly but with little or no selectivity. This is perhaps
unsurprising considering the lack of steric discrimination available
to the enzyme on the tetrahydrofuran ring system. No literature
data on the resolution of compound 5a could be found. This com-
pound has been synthesized in 81% yield and 76 % ee by coupling
4-chlorophenylboronic acid to benzaldehyde using diethylzinc in
Figure 1 depicts the substrates chosen to test the ability of the li-
pase panel to successfully resolve compounds that are not currently
⇑
Corresponding author.
E-mail address: david.mangan@almacgroup.com (D. Mangan).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.03.013

584
M. O’Neill et al. / Tetrahedron: Asymmetry 23 (2012) 583–586
Table 1
Table 2
Literature data on the CALB resolution of the assembled selection of secondary
alcohols
Selected screening results from the lipase screen of compounds 1a–5a
Compound Conditions
Selectivity
(R)/(S) (ester)
30 °C, 163 h (R)
Conversion (%)
O
E
OH
vinyl acetate
lipase
O
OH
1aa
2aa
3aa
4ab
5ac
19.4
17.3 (1 mmol sub;
200 mg CALB)
5.9 (1 mmol sub;
200 mg CALB)
33.7 (1 mmol sub;
200 mg CALB)
(1.5 mmol sub;
25 mg CALB)
R1
R2
R1
R2
R1
R2
30 °C, 168 h (R)
30 °C, 96 h (R)
37 °C, 5 min (S)
N/A
65.1
9.0
3.0
Compound
Enzyme
number
Time
(h)
Results
Alcohol
Ester
(% ee)
Conversion
(%)
E
(R)/(S) depending on the N/A
ligand, with ee up to
75.8%
(% ee)
1a
EL-01
EL-03
EL-13
EL-14
EL-68
CALB
EL-01
EL-70
CALB
EL-01
EL-12
EL-70
CALB
EL-13
EL-14
EL-15
CALB
EL-01
EL-12
EL-13
EL-14
EL-68
CALB
101
101
101
101
101
101
16
16
16
16
16
16
16
0.08
1
1
11
11
3
2
10
3
23
30
<1
26
2
34
27
2
7
12
8
3
3
3
3
3
N/A
97
98
91
90
92
11
10
3
30
110
21
19
26
Reported.14
a
b
c
Reported.15
2
No literature precedent with CALB. Synthesised using chiral organozinc
10
<1
22
27
1
43
2
51
25
5
reagents.21
>99
>200
>200
>200
32
2a
3a
>99
99
94
33
84
33
79
40
48
47
52
27
53
2
the presence of 20 mol % of chiral amino alcohols derived from cis-
(1R,2S)-2-benzamidocyclohexanecarboxylic acid. However, this is
not an atom efficient process and lacks economical applicability
on a larger scale.
2.5
11
2.7
11
4a
5a
2.5
3.3
3.1
3.4
1.8
3.4
1.1
1.0
1.9
N/A
2. Results and discussion
15
21
14
1
1
In order to assess the enantioselectivity obtained in the enzy-
matic screening reactions, racemic standards of the acetylated
alcohols 1b–5b were prepared using established chemistry. Ana-
lytical methods (chiral HPLC/GC) were developed based on the
characteristics of each compound and are discussed in detail in
the Experimental. Selected results from the 23 lipase enzyme
screen are outlined in Table 2.
101
101
101
101
87
5
60
75
9
1
30
101
N/A
<1
The initial screening results were extremely encouraging. The
stereopreference of the novel lipase enzymes for each substrate
tested was found to be the same as that exhibited by CALB. How-
ever, a number of distinct lead enzymes emerged as being more
active and/or more selective catalysts for a number of these
substrates.
For compound 1a, EL-01, EL-03, and EL-68 exhibited excellent
activity versus the CALB control but while the use of EL-01 and
EL-68 resulted in only a modest selectivity factor, EL-03 appeared
to also facilitate excellent selectivity (E ꢀ110) for the desired
bioresolution.
In the same manner, EL-01 and EL-70 served to acetylate com-
pound 2a with excellent selectivity and activity when compared to
the CALB control. The results from the lipase screen on compound
3a were somewhat less conclusive with no lipase found that pos-
sessed the desired level of activity and selectivity. It was thought
that following reaction optimization, EL-12 might prove to be a
successful candidate for the desired transformation.
Compounds 4a and 5a were expected to be the most challeng-
ing of the substrates screened. Compound 4a, possessing a com-
pletely unhindered alcohol moiety was overacylated in our initial
screening experiments, which were allowed to react for 4 h. A
reduction in the reaction time to one hour allowed the extraction
of genuine selectivity factor data (<50% conversion) on the reac-
tions in question. Although EL-14 displayed selectivity that was be-
low a synthetically useful level, this result was still marginally
superior to the analogous reaction mediated by CALB.
of this reaction. The CALB control experiment produced only trace
levels of racemic ester.
It has been well documented that the presence of organic co-
solvents can have a large effect on the activity and selectivity of
enzymatic processes.16–21 As a result, we thought that a brief
investigation into the tolerance of this lipase enzyme panel to
the most commonly used organic co-solvents was warranted. The
lead enzyme from each of the acetylation reactions on compounds
1a–5a was screened in the presence of hexane, MTBE, 2-methyl-
THF, and toluene. CALB control reactions were carried out in paral-
lel. The results are outlined in Table 3.
As was expected, the organic co-solvents screened against each
lead enzyme produced a broad range of results. MtBE appears to be
the most generally applicable organic co-solvent for reactions of
this type. This reaffirmed our choice of MtBE as the co-solvent
for all initial enzyme screens but the value of subsequent co-sol-
vent screening is also highlighted in these results. For the conver-
sion of 3a to 3b, hexane produces a notable increase in conversion
with EL-12 compared to the analogous MTBE reaction while hex-
ane produces a significant increase in selectivity (ꢀ50%) with CALB
when compared to the analogous reaction with MtBE. Similarly, for
the conversion of 5a to 5b, MeTHF produces a significant increase
in selectivity (albeit with a marked loss in activity) with both EL-12
and CALB when compared to the analogous reactions with MtBE.
Interesting results were obtained from the lipase screening of
compound 5a. Extreme diversity in this lipase panel was evident
from the fact that EL-13 and EL-14 displayed high, non-selective
activity for the acetylation of compound 5a, while EL-12 exhibited
mild selectivity but produced only 5% conversion over the course
3. Conclusion
A number of secondary alcohols that are known to be difficult to
obtain in enantioenriched form from their racemates via enzymatic
kinetic resolution have been screened against 23 novel lipase

M. O’Neill et al. / Tetrahedron: Asymmetry 23 (2012) 583–586
585
Table 3
enantiomeric excess measurements of compounds 1a–4a and
1b–4b were determined by GC analysis using a PerkinElmer Auto-
System XLGC.
Co-solvent screening results for the enzymatic acetylation of 1a–5a to 1b–5b,
respectively, using lead enzymes identified from the initial screening
Enzyme
number
Time
(h)
Solvent
Results
For 1-phenylbutan-1-ol 1a, analysis was performed on a Varian
Chirasil DEX CB column (25 m  0.25 mm  0.25
lm). Helium was
Alcohol
(% ee)
Ester
(% ee)
Conversion
(%)
E
used as a carrier gas. Method: 100 °C hold for 40 min, 5 °C minÀ1
until 180 °C, hold for 3 min; inlet temperature 250 °C; detector
temperature 280 °C; flow rate: 3.4 mL minÀ1. Retention times for
(R)-1a and (S)-1a were 38.9 and 40.2 min, respectively. Retention
times for (S)-1b and (R)-1b were 19.4 and 19.7 min, respectively.
The assignment of the absolute stereochemistry for each enantio-
mer was achieved by comparison with the literature data for the
analogous CALB mediated reaction.14
1a
2a
3a
4aa
5a
EL-03
CALB
EL-70
CALB
EL-12
CALB
EL-14
CALB
EL-12
CALB
16
16
16
16
16
16
16
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
14
14
92
98
3
4
24
103
No reaction
10
9
88
87
98
1
4
2
15
14
100
18
No reaction
10
8
36
<1
6
<1
<1
97
3
11
31
<1
8
67
73
152
2.5
71
97
98
43
97
84
82
For 1-cyclohexyl-1-butanol 2a, analysis was performed on a
Varian Chirasil DEX CB column (25 m  0.25 mm  0.25
lm). He-
lium was used as a carrier gas. Method: 80 °C hold for 3 min,
1 °C minÀ1 until 130 °C, 10 °C minÀ1 until 180 °C, hold for 3 min;
inlet temperature 250 °C; detector temperature 280 °C; flow rate:
3.4 mL minÀ1. Retention times for (S)-2a and (R)-2a were 33.2
and 34.3 min, respectively. Retention times for (S)-2b and (R)-2b
were 29.3 and 30.0 min, respectively. The assignment of the abso-
lute stereochemistry for each enantiomer was achieved by com-
parison with the literature data for the analogous CALB mediated
reaction.14
1
1
11
10
No Reaction
<1
6
68
20
77
<1
24
3
5.2
1.6
7.9
2
No Reaction
5
13
11
No Reaction
8
41
8
1
6
9
7
6
8
4
2.5
1
1
24
86
81
17
13
12
1.7
15
10
For 4-decanol 3a, analysis was performed on a Varian Chirasil
81
7
46
14
4
12
29
19
17
16
8
6
1
8
13
17
4
10
DEX CB column (25 m  0.25 mm  0.25
lm). Helium was used
as a carrier gas. Method: 80 °C hold for 3 min, 1 °C minÀ1 until
46
52
16
45
23
31
30
40
48
45
72
12
20
10
52
4.0
3.4
1.4
2.8
1.7
2.0
2.0
2.5
3.0
2.7
6.2
1.3
1.5
1.2
3.2
130 °C, 10 °C minÀ1 until 180 °C hold for 3 min; inlet temperature
250 °C; detector temperature 280 °C; flow rate: 3.4 mL minÀ1
.
Retention time for 3a was 23.9 min. Retention times for (S)-3b
and (R)-3b were 22.6 and 23.6 min, respectively. The enantiomeric
excess of 3a was determined theoretically from reaction conver-
sion and the ee measured for 3b. The assignment of the absolute
stereochemistry for each enantiomer was achieved by comparison
with the literature data for the analogous CALB mediated
reaction.14
1
1
16
1
40
Me-THF
Toluene
Hexane
MTBE
Me-THF
Toluene
Hexane
MTBE
40
3
2
2
For 3-hydroxytetrahydrofuran 4a, analysis was performed on a
Supelco b-DEX 225 column (30 m  0.25 mm  0.25
lm). Helium
Me-THF
Toluene
was used as a carrier gas. Method: 80 °C hold for 3 min, 5 °C minÀ1
until 180 °C, hold for 10 min; inlet temperature 250 °C; detector
temperature 280 °C; flow rate: 3.4 mL minÀ1. Retention times for
(S)-4a and (R)-4a were 13.2 and 13.5 min, respectively. For tetra-
hydrofuran-3-yl acetate 4b, analysis was performed on a Varian
No Reaction
a
Reactions with 4a were carried out at 4 °C. All other reactions were carried out
at 30 °C.
enzymes. Following initial enzyme screening using MtBE as the
organic co-solvent, a lead enzyme was selected for each desired
transformation and a small co-solvent screen was performed with
2-MeTHF, toluene, and hexane, along with a control reaction in
MtBE. CALB was used throughout this entire study as a benchmark
for activity and selectivity. For all five compounds 1a–5a screened
at least one novel lipase from the panel was found to outperform
CALB in terms of activity and/or selectivity.
Chirasil DEX CB column (25 m  0.25 mm  0.25
lm). Helium
was used as a carrier gas. Method: 80 °C hold for 3 min, 5 °C minÀ1
until 140 °C, 10 °C minÀ1 until 180 °C, hold for 3 min; inlet temper-
ature 250 °C; detector temperature 280 °C; flow rate:
3.4 mL minÀ1. Retention times for (R)-4b and (S)-4b were 7.0 and
7.3 min, respectively. The assignment of the absolute stereochem-
istry for each enantiomer was achieved by comparison with the lit-
erature data for the analogous CALB mediated reaction.15
Enantiomeric
phenyl)(phenyl)methanol 5a were determined by normal phase
m, Daicel
Chemical Industries) with UV detection (k, 254 nm), eluent hex-
ane/ethanol (95:5); flow rate 0.7 mL/min. Retention times for (R)-
5a and (S)-5a were 13.5 and 14.3 min, respectively. Enantiomeric
excess measurements of 5b were determined by normal phase
excess
measurements
of
(4-chloro-
4. Experimental
HPLC on a Chiralcel IA column (250 mm  4.6 mm  10
l
4.1. Chemicals and enzymes
The novel lipase enzymes discussed herein are commercially
available from both Almac and Eucodis Bioscience GmbH.
Chemicals were purchased from Sigma Aldrich, Alfa Aesar and
TCI. Lipase enzyme CALB (Novozym 435) was obtained from
Enzagen Ltd.
HPLC on a Chiralcel OJ column (250 mm  4.6 mm  10
lm, Daicel
Chemical Industries) with UV detection (k, 254 nm), eluent hex-
ane/ethanol (95:5); flow rate 1.0 mL/min. Retention times for (S)-
5b and (R)-5b esters were 9.27 and 10.42 min, respectively. It
should be noted that although routine screening analysis was car-
ried out on the IA and OJ columns as these provided excellent peak
resolution, the assignment of the absolute stereochemistry for each
enantiomer was achieved by comparison with the literature data
4.2. Analytical methods
1H NMR spectra were recorded at 400 MHz on a Bruker AV-400
spectrometer; shifts are relative to internal TMS. Conversion and

586
M. O’Neill et al. / Tetrahedron: Asymmetry 23 (2012) 583–586
for the analogous CALB mediated reaction, using a Chiralpak AD-H
column.16
4.3. General enzyme screening conditions
Lipase enzyme lyophilized powder (2 mg), was added to a solu-
4.2.1. ( )-1-Acetoxy-1-phenylbutane 1b
tion of the substrate (13.3
followed by addition of a solution of vinyl acetate (66.5
l
mol) in the reaction solvent (0.5 mL),
mol) in
To a stirring solution of 4-dimethylaminopyridine (81 mg,
0.66 mmol) in dichloromethane (5 mL) was added triethylamine
(0.51 mL, 3.66 mmol) followed by addition of 1-phenylbutan-1-ol
(1a, 0.50 g, 3.33 mmol). Acetic anhydride (0.38 mL, 3.99 mmol)
was added dropwise and the resultant solution was stirred at room
temperature for two hours. The reaction mixture was washed with
10% HCl (2 Â 5 mL) and 1 M NaHCO3 (5 mL) and the organic layer
was dried over anhydrous MgSO4 and concentrated in vacuo to af-
ford a clear oil (0.40 g, 63%). dH (400 MHz): 0.91 (3H, t, J = 7.6),
1.19–1.42 (2H, m), 1.69–1.78 (1H, m), 1.85–1.94 (1H, m), 2.07
(3H, s), 5.73 (1H, dd, J = 7.6 and 6.3), 7.26–7.36 (5H, m).
l
the reaction solvent (0.5 mL). The reactions were shaken at
150 rpm at 30 °C for an appropriate amount of time. The reactions
were filtered through anhydrous magnesium sulfate and analyzed
directly by chiral GC or evaporated, redissolved in iso-propyl alco-
hol (HPLC grade) and analyzed by chiral HPLC. The screening re-
sults are summarized in Tables 2 and 3.
Acknowledgments
This study was part financed by the European Regional Devel-
opment Fund under the European Sustainable Competitiveness
Programme for Northern Ireland. Lipase library generation by
EUCODIS Bioscience was part financed by the Austrian Research
Promotion Agency, Vienna.
4.2.2. ( )-1-Cyclohexylbutyl acetate 2b
The title compound was prepared following the procedure for
the preparation of ( )-1-acetoxy-1-phenylbutane 1b, from 4-
dimethylaminopyridine (31 mg, 0.26 mmol), triethylamine
(0.24 mL, 0.18 mmol), 1-cyclohexyl-1-butanol 2a (0.20 g,
0.13 mmol), and acetic anhydride (0.15 mL, 0.15 mmol) to afford
a clear oil (156 mg, 61 %). dH (400 MHz): 0.90 (3H, K, J = 7.0),
0.94–1.05 (2H, m), 1.10–1.37 (5H, m), 1.42–1.52 (3H, m), 1.63–
1.76 (5H, m), 2.05 (3H, s), 4.75 (1H, dd, J = 12.4 and 7.0).
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4.2.5. ( )-(4-Chlorophenyl)(phenyl)methyl acetate 5b
The title compound was prepared following the procedure for
the preparation of ( )-1-acetoxy-1-phenylbutane 1b, from 4-
dimethylaminopyridine (381 mg, 0.67 mmol), triethylamine
(0.51 mL, 3.67 mmol), 4-chlorobenzohydrol 5a (0.73 g, 3.33 mmol),
and acetic anhydride (0.38 mL, 3.99 mmol) to afford a clear oil
(516 mg, 60 %). dH (400 MHz): 2.16 (3H, s), 6.84 (1H, s), 7.26–
7.37 (9H, m).