Influence of the position of the substituent on the efficiency of lipase-mediated resolutions of 3-aryl alkanoic acids

Article abstract of DOI:10.1016/j.tetasy.2013.09.019

Hydrolase-catalysed kinetic resolutions to provide enantioenriched α-substituted 3-aryl alkanoic acids are described. (S)-2-Methyl-3- phenylpropanoic acid (S)-1a was prepared in 96% ee by Pseudomonas fluorescens catalysed ester hydrolysis, while, Candida antarctica lipase B (immob) resolved the α-ethyl substituted 3-arylalkanoic acid (R)-1b in 82% ee. The influence of the position of the substituent relative to the ester site on the efficiency and enantioselectivity of the biotransformation is also explored; the same lipases were found to resolve both the α- and β-substituted alkanoic acids. Furthermore, the steric effect of substituents at the C2 stereogenic centre relative to that for their C3 substituted counterparts on the efficiency and stereoselectivity is discussed.

Full text of DOI:10.1016/j.tetasy.2013.09.019

Tetrahedron: Asymmetry 24 (2013) 1480–1487
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Influence of the position of the substituent on the efficiency
of lipase-mediated resolutions of 3-aryl alkanoic acids
Rebecca E. Deasy a, Thomas S. Moody b, Anita R. Maguire a,
⇑
a Department of Chemistry & School of Pharmacy, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
b Department of Biocatalysis & Isotope Chemistry, Almac, Craigavon BT63 5QD, Northern Ireland, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrolase-catalysed kinetic resolutions to provide enantioenriched
are described. (S)-2-Methyl-3-phenylpropanoic acid (S)-1a was prepared in 96% ee by Pseudomonas
fluorescens catalysed ester hydrolysis, while, Candida antarctica lipase B (immob) resolved the -ethyl
substituted 3-arylalkanoic acid (R)-1b in 82% ee. The influence of the position of the substituent relative
a-substituted 3-aryl alkanoic acids
Received 17 August 2013
Accepted 19 September 2013
Available online 29 October 2013
a
to the ester site on the efficiency and enantioselectivity of the biotransformation is also explored; the
same lipases were found to resolve both the a- and b-substituted alkanoic acids. Furthermore, the steric
effect of substituents at the C2 stereogenic centre relative to that for their C3 substituted counterparts on
the efficiency and stereoselectivity is discussed.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and electronic effects on the efficiency and enantioselectivity of
the biocatalytic transformation were also explored.
The use of hydrolases in kinetic resolution has grown enor-
mously in popularity, from an academic curiosity a century ago
Leading on from the success of this lipase-mediated hydrolysis
study, we extended this work to a series of 3-aryl alkanoic acids
to
a
transformation that is routinely used in industry.1–3
that were alkylated
a to the ester moiety. Unlike our earlier study
These environmentally benign, biodegradable catalysts are effec-
tive under mild reaction conditions, and combine wide substrate
specificity with high regio- and enantioselectivity enabling the
resolution of organic substrates with superb efficiency.4–6
We have recently reported on successful hydrolase-catalysed
kinetic resolutions leading to a series of highly enantioenriched
3-aryl alkanoic acids through optimisation of the reaction condi-
tions (Scheme 1).7 Hydrolysis of the ethyl esters with a series of
hydrolases was undertaken and it was found that through an
appropriate choice of biocatalyst and careful control of the reaction
conditions, the corresponding b-substituted acids were formed
with excellent enantiopurity in each case (P94% ee). The steric
on the b-substituted series,7 the chiral resolution of carboxylic
acids with
a stereogenic centre at the a-position has been
examined extensively.8–10 In particular, the literature has been
dominated by the successful resolution of commercially important
2-aryl or 2-aryloxypropionic acids, the former are non-steroidal
anti-inflammatory drugs while the latter are an important class
of herbicides.11–17
Enantiopure
a-substituted 3-aryl alkanoic acids are attractive
synthetic targets18 and traditional aqueous hydrolase-catalysed
ester hydrolysis has been previously described for the resolution
of 2-methyl-3-phenylpropanoic acid (S)-1a with excellent
enantioselectivity being achieved (95% ee).19 However, it has been
R1
O
R1
*
O
R1
*
O
hydrolase
OEt
OH
OEt
X
X
X
≥94% ee
Scheme 1. Lipase-catalysed kinetic resolutions of b-substituted 3-aryl alkanoic acids.7
⇑
Corresponding author. Tel.: +353 21 490 1694/2125/3501; fax: +353 21
4901770.
E-mail address: a.maguire@ucc.ie (A.R. Maguire).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.09.019

R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
1481
demonstrated that the hydrolysis of acid substrates encompassing
more sterically demanding substituents at the C2 stereogenic cen-
tre is much less successful.20 In fact, a dramatic reduction of activ-
ity and enantioselectivity was reported in the lipase-mediated
resolution of 2-benzylbutanoic acid (S)-1b (53% ee), in comparison
to 1a, upon replacement of the methyl with the bulkier ethyl
substituent.21
Herein we report our studies exploring a range of alkyl substit-
uents at the C2 stereogenic centre and examining the effect of the
position of the substituent relative to the active site on the effi-
ciency of the bioresolution. The steric effect of the substituents at
C2 relative to that previously reported for their C3 substituted
counterparts upon enantioselection was also examined. The C2
substituted methyl, ethyl and tert-butyl 3-arylalkanoic acids 1a–c
were selected for investigation (Scheme 2).
the (S)-enantiomer of ethyl 2-methyl-3-phenylpropanaote 2a.
Margolin et al. previously reported the Pseudomonas sp. (Amano)
mediated hydrolysis of the corresponding methyl ester of 1a pro-
viding the acid (S)-1a with high enantiomeric excess (95% ee).19
It should be noted Margolin’s study was limited to the screening
of the lipase Pseudomonas sp. (Amano), while a broad series of
lipases were examined herein which identified Alcaligenes spp. 2
in addition to Pseudomonas cepacia P2 and Pseudomonas fluorescens
for the enzymatic-catalysed hydrolysis of 2a providing highly
enantioenriched (S)-1a.
The Pseudomonas lipases afforded the (S)-acid 1a (P92% ee) and
(R)-ester 2a (>98% ee) in excellent enantiopurity (Table 1, entries
10a and 13a). Alcaligenes spp. 2 generated the (S)-acid 1a with an
improved enantioselectivity of 97% ee (Table 1, entry 9). However
the rate of the resolution was decreased (conversion 41%) and
therefore the enantiopurity of the recovered (R)-ester 2a (67% ee)
was compromised.
2. Results and discussion
The Candida cyclindracea C2 and Candida antarctica lipase B (free
and immobilised) (Table 1, entries 2, 14 and 16, respectively) med-
iated hydrolysis proceeded with 100% conversion to racemic acid
1a exhibiting a lack of discrimination between the enantiomers.
Bornscheuer and Kazlauskas reported that Candida antarctica
lipase B usually displays low to moderate enantioselectivity to-
2.1. Synthesis of ethyl 3-aryl alkanoates
The 3-arylalkanoic acids 1a and 1c were commercially available
and esterification yielded the ethyl 3-arylalkanoates 2a and 2c
which were obtained as substrates in moderate yield (63% and
53%, respectively). Racemic ester 2a was obtained via a Fischer
esterification reaction while an alternative SN2 approach employ-
ing potassium carbonate and ethyl iodide was adopted for the
esterification of the sterically hindered 1c (Scheme 3). The 3-aryl
alkanoic acid 1b was not commercially accessible and thus 2b
wards carboxylic acids with a stereocentre at the
a
-position.5
The acyl binding site of Candida antarctica lipase B is a shallow
crevice. It is likely that the lower enantioselectivity towards stereo-
centres in the acyl part of an ester stems from fewer and/or weaker
contacts between the acyl part and its binding site.
Significantly, Alcaligenes spp. 2 and the Pseudomonas lipases
were also identified to yield the highest enantiopurity upon resolu-
tion of the structurally related b-substituted 3-phenylbutanoic acid
(S)-1d (P94% ee) and the analogous ethyl ester (R)-2d (>98% ee)
albeit at extended reaction times (65 h) (Scheme 4).7 Thus, the
position of the chiral methyl substituent relative to the reactive
ester moiety has a limited effect on the choice of biocatalyst or
high enantiopurity obtainable; however, the reaction rate is
altered with efficient resolution achieved within 20 h for (S)-1a
versus 65 h for (S)-1d.
was synthesised via a-alkylation of ethyl butyrate employing lith-
ium diisopropylamide and benzyl bromide (50% yield) (Scheme 3).
With racemic samples of both the esters 2a–c and acids 1a–c in
hand, chiral HPLC conditions were developed to enable determina-
tion of the enantiopurity of the substrate and product through a
single injection of the reaction mixture.
2.2. Hydrolase-catalysed kinetic resolution to provide
enantioenriched 2-methyl-3-phenylpropanoic acid 1a
Based on the screening results in Table 1, the use of Pseudomo-
nas cepacia P2 and Pseudomonas fluorescens (entries 10a and 13a,
respectively) was evidently the most attractive from the
perspective of preparing enantioenriched samples of the acid
In total, 18 lipases were screened in the lipase-catalysed kinetic
resolution to provide enantioenriched 2-methyl-3-phenylpropa-
noic acid 1a as summarised in Table 1. All of the enzymatic
resolutions investigated resulted in at least partial hydrolysis of
O
O
O
*
*
hydrolase
OEt
OH
OEt
R1
R1
R1
2a R1 = CH3
1a R1 = CH3
2a-c
1 = CH2CH3
1 = CH2CH3
1b
R
2b
R
2c R1 = C(CH3)3
1c R1 = C(CH3)3
Scheme 2. Lipase-catalysed kinetic resolutions of
a
-substituted 3-aryl alkanoic acids.
A:
H2SO4, EtOH
or
C:
O
O
O
i) LDA, -78 °C
OEt
B:
K2CO3, CH3CH2I, C3H6O
OH
OEt
ii) C6H5CH2Br
R1
R1
iii) H3O+
1 = CH3
1c R1 = C(CH3)3
R
1 = CH3
2b R1 = CH2CH3 C: 50% yield
2c B:
63% yield
1a
R
2a
A:
R
1 = C(CH3)3
53% yield
Scheme 3. Synthesis of ethyl 3-aryl alkanoates 2a–2c.
The bacterium Pseudomonas cepacia is now known as Burkholderia cepacia.

1482
R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
Table 1
Hydrolase-mediated hydrolysis of ethyl 2-methyl-3-phenylpropanoate 2a
O
O
O
hydrolase
OEt
OH
OEt
0.1 M Phosphate buffer, pH 7.0
RT
( )-2a
1a
(S)-
2a
(R)-
Entry
Enzyme source
Time (h)
eea (%)
Conversionb (%)
E valueb
Ester (R)-2a
Acid (S)-1a
1
2
3
4
5
6
7
8
Candida cyclindracea C1
Candida cyclindracea C2
Alcaligenes spp. 1
Pseudomonas cepacia P1
Pseudomonas stutzeri
Rhizopus spp.
Rhizopus niveus
Aspergillus niger
Alcaligenes spp. 2
Pseudomonas cepacia P2
20
20
72
72
72
72
72
72
20
20
10
20
72
20
10
20
20
20
72
72
3c
3c
50
1.1
0d
0d
Complete conversion
15
44
62
14
49
13
41
52
50
40
15
52
46
2
11
68
50
30
21
33
97
93
96
21
17
92
97
0d
1.3
6.4
17
1.9
1.8
53
83
5
20
5
67
>98
95
14
3
>98
81
0d
2.1
9
132
>200
183
1.7
1.5
179
164
10a
10b
11
12
13a
13b
14
15
16
17
18
Mucor javanicus
Penicillium camembertii
Pseudomonas fluorescens
Candida antarctica B
Mucor meihei
Candida antarctica B (immob)
Porcine pancrease Type II
Porcine pancrease Grade II
Complete conversion
52
Complete conversion
39
19
23
0
1.9
0d
0d
58
11
90
47
34
3.1
a
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, Step gradient: 45 °C, injection volume 2 lL, k = 209.8 nm, hexane/i-PrOH (3% trifluoroacetic acid), 0–49 min;
99.5:0.5, flow rate 0.2 mL/min, 50 min; 95:5, flow rate 1.0 mL/min].
b
Conversion and the enantiomeric excess (E value) was calculated from the enantiomeric excess of substrate ester 2a (ees) and product acid 1a (eep).22
Limited enantiopurity observed, thus the direction of enantioselection should be interpreted with caution.
c
d
Reaction went to 100% completion (conversion determined by 1H NMR), no enantioselectivity observed.
O
O
O
Alcaligenes spp. 2 or
Pseudomonas cepacia P1
OH
OEt
OEt
or Pseudomonas fluorescens
65 h
( )-2d
1d
(S)-
(≥94% ee)
2d
(R)- (>98% ee)
Scheme 4. Hydrolase-mediated hydrolysis of ethyl 2-methyl-3-phenylpropanoate 2d.7
(S)-1a. It is clear from the extent of conversion (>50%) and the
slightly low enantiopurity of the acid (S)-1a (92 and 93% ee) that
a small amount of the ester (R)-2a undergoes hydrolysis during
the kinetic resolution. Accordingly, kinetic resolutions were under-
taken with a shorter reaction time of 10 h, under otherwise identi-
cal reaction conditions, and significantly, as we anticipated, the
enantiopurity of the recovered acids (S)-1a was enhanced to 96%
and 97% ee (entries 10b and 13b, respectively). This observation
highlights that with optimisation, highly enantioenriched samples
of (S)-1a could be obtained.
In order to demonstrate the practical viability of this process,
Pseudomonas fluorescens was selected as the most suitable hydro-
lase for the preparative scale (1.21 mmol) hydrolysis of ethyl
2-methyl-3-phenylpropanaote 2a. The conversion and enantiopu-
rity of ester 2a and acid 1a were analysed by utilising chiral HPLC
and an optimum 50% conversion was achieved with both enantio-
mers being produced in P96% ee. The highly enantioenriched acid
(S)-1a (96% ee) was isolated in 37% yield after column chromatog-
raphy and the ester (R)-2a (>98% ee) was recovered in 27% yield
(Table 2, entry 1).à
2.3. Hydrolase-catalysed kinetic resolution to provide
enantioenriched 2-benzylbutanoic acid 1b
On increasing the size of the methyl moiety at the C2 site to the
bulkier ethyl group, a sharp decrease in both efficiency and enan-
tioselection was observed, with a number of hydrolases displaying
no hydrolysis. This correlated strongly with the literature
reports20,21,26 and with the trends observed with the C3
substituted 3-arylalkanoic acids,7 demonstrating the dramatic
dependence of the synthetic and stereochemical outcome of the
reaction upon the size of the alkyl group at the stereogenic centre.
Herein Candida antarctica lipase B (Table 3, entry 3) provided
the highest enantiopurity of 2-benzylbutanoic acid (R)-1b (83%
ee) via lipase-mediated hydrolysis. Previous to this result, the high-
est reported lipase-mediated resolution of (S)-1b was 53% ee.21
Herein Candida antarctica lipase B (free and immobilised) provided
the (R)-enantiomer selectively, while all of the other reported
hydrolases preferentially hydrolysed the (S)-enantiomer, albeit
with low to modest enantioselectivity.
The decreased efficiency in the kinetic resolution in the Candida
antarctica lipase B mediated hydrolysis of 2b relative to 2a is nota-
ble. This may be due to the fact that in both the ethyl and benzyl
substituents of 2b, a methylene group is adjacent to the stereogen-
ic centre, with discrimination of the groups only at the next carbon.
à
Yields may be reduced due to reaction sampling.

R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
1483
Table 2
Synthetic scale hydrolase-mediated kinetic resolution of C2 substituted alkanoic esters 2a–b
O
O
O
hydrolase
*
*
OEt
OH
OEt
0.1 M Phosphate buffer, pH 7.0
R
R
R
Entry
1
R
Hydrolase
Conversiona (%)
51
Acid
Ester
Yield (%)
ee (%)
Specific rotationb
37c
27c
96
½
½
a 2D0
a 2D0
ꢀ
¼ þ28:0 (c 0.82, CHCl3)23
Me
P. fluorescens
(S)-1a
ꢀ
¼ ꢁ36:4 (c 1.0, CHCl3)24
(R)-2a
>98
a 2D0
ꢀ
¼ ꢁ43:8 (c 1.0, CH2Cl2)25
2
Et
Candida antarctica B (immob)
24
(R)-1b
19
43
82
26
½
½
a 2D0
ꢀ
¼ þ6:8 (c 1.0, CH2Cl2)d
(S)-2b
a
Conversion was calculated from the enantiomeric excess of substrate ester 2a or 2b (ees) and product acid 1a or 1b (eep).22
b
The direction of enantioselection of each of the acids (S)-1a23 and (R)-1b25 and recovered ester (R)-2a24 was determined by comparing the specific rotation data obtained
in this study with those reported in the literature.
c
Yield may be reduced due to reaction sampling.
Although the a-ethyl substituted ester (S)-2b has not previously been reported in its enantioenriched form, its absolute stereochemistry was assigned as (S) as it must be
d
opposite to that of the recovered acid (R)-1b.
Table 3
Hydrolase-mediated hydrolysis of ethyl 2-benzylbutanoate 2b
O
O
O
hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7.0
RT
2b
( )-
Enzyme sourcea
1b
2b
(S)-
(R)-
Entry
Time (h)
eeb (%)
Conversionc (%)
E valuec
Ester 2b
Acid 1b
1
2
3
4
5
6
Candida cyclindracea C1
Candida cyclindracea C2
Candida antarctica B
Candida antarctica B
Candida antarctica B (immob)
Pig Liver esterase
43
17
17
43
43
17
3 (R)
13 (S)
4 (S)
83 (R)
71 (R)
73 (R)
3 (S)d
19
83
30
49
19
67
1.3
1.3
15
14
7.6
1.1
20 (R)
35 (S)
74 (S)
17 (S)
6 (R)d
a
The following hydrolases gave no conversion Pseudomonas cepacia P2, Pseudomonas cepacia P1, Mucor javanicus, Pseudomonas fluorescens, Porcine Pancrease Type II,
Pseudomonas stutzeri, Rhizopus niveus and Penicillium camembertii.
b
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 95:5, flow rate 0.5 mL/min, ambient temperature, injection volume
2
l
L, k = 209.8 nm].
c
Conversion and the enantiomeric excess (E value) was calculated from the enantiomeric excess of substrate ester 2b (ees) and product acid 1b (eep).22
Limited enantiopurity was observed, thus the direction of enantioselection should be interpreted with caution.
d
It should be noted that 4 of the 5 hydrolases screened which
herein for (R)-1b (83% ee) (Table 3, entry 3). Thus, the position of
the ethyl moiety had a limited impact on the choice of the
biocatalyst.
Herein the enantiomeric excess of the unreacted ester (S)-2b
was poor (35% ee) (Table 3, entry 3) relative to that of the b-substi-
tuted analogue (S)-2e (85% ee) (Scheme 5), due to the decreased
conversion rate (30% vs 51%). Increasing the reaction time from
17 h to 43 h (Table 3, entry 4) did result in a higher conversion rate
(49%) and increased the enantiomeric purity of (S)-2b (74% ee),
resulted in conversion (Table 3), also displayed enantioselection
for the resolution of the b-ethyl substituted 3-phenylpentanoic
acid 1e, the exception being Candida cylindracea C1 which recorded
no catalytic activity for the hydrolysis of 2e.7 In addition, under
similar reaction conditions, the same source of enzyme Candida
antarctica lipase B, although the immobilised version rather than
the free enzyme, afforded the highest enantiopurity of (R)-1e
(81% ee) (Scheme 5) comparable to the enantioselectivity obtained
O
O
O
Candida antarctica B (immob)
OH
OEt
OEt
65 h
(
2e (
85% ee)
S)-
1e
(R)- (81% ee)
( )-2e
Scheme 5. Hydrolase-mediated hydrolysis of ethyl 3-phenylpentanoate 2e.7

1484
R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
however the enantiopurity of the acid (R)-1b was compromised
(71% ee) due to the partial hydrolysis of ester (S)-2b at the
extended reaction time.
3. Conclusion
While -substituted phenyl propanoic acids can be resolved
a
Thus, the enantiodiscrimination in the hydrolysis to form the
using a lipase-mediated kinetic resolution, the outcome in terms
a-ethyl acid (R)-1b was somewhat less efficient than that in the
of both efficiency and selectivity is strongly dependent upon the
corresponding b-ethyl acid (R)-1e despite the increased proximity
of the stereocentre. This may again be due to the fact that in both
the ethyl and benzyl substituents, a methylene group is adjacent
to the stereogenic centre, with discrimination of the groups only
at the next carbon.
The synthetic scale (1.95 mmol) hydrolysis of ethyl 2-benzyl-
butanoate 2b was performed next. In the analytical screens, free
Candida antarctica lipase B resulted in the highest enantioselectiv-
steric features of the a-substituent. Thus, when the a-substituent
increased in size from a methyl to an ethyl or tert-butyl group, a
dramatic decrease in the rate of conversion and enantioselectivity
of the hydrolysis was recorded. This correlated with earlier reports
in the literature20,21,26 and with the results observed with the
b-substituted 3-arylalkanoic acids.7 Despite the steric hindrance
within the active site, (S)-2-methyl-3-phenylpropanoic acid
(S)-1a was obtained in 97% ee, via Alcaligenes spp. 2 catalysed
hydrolysis of 2a while Candida antarctica lipase B was identified
ity (83% ee) of the sterically hindered a-ethyl acid (R)-1b (Table 3,
entry 3). However, Candida antarctica lipase B (immob) was
utilised in the preparative-scale since immobilised lipases offer
significant advantages over their free counterparts from the
perspective of large-scale process efficiency.5 Herein the Candida
antarctica lipase B (immob) mediated resolution of 2b on a pre-
parative-scale resulted in a slight increase in conversion rate
(24% vs 19%) and thus improved the enantiopurity of (R)-1b
(82% ee) and (S)-2b (26% ee) relative to that from the small scale
screen (73% ee and 17% ee, respectively) (Table 2, entry 2 vs
Table 3, entry 5).
as resolving the
a-ethyl substituted 3-arylalkanoic acid (R)-1b
(82% ee) with improved enantioselection relative to that achieved
by Sih et al. (53% ee)21 (Fig. 1). Significantly we have demonstrated
that through variation of reaction time, the enantiopurity of the
recovered acids (S)-1a and (R)-1b can be optimised. Thus these bio-
transformations have the potential to be synthetically useful pro-
cesses since the hydrolysis of the slower reacting enantiomer of
the esters (R)-2a and (S)-2b can be minimised by careful reaction
control. Furthermore, the choice of biocatalyst was determined to
be independent of the position of the substituent relative to the
reactive site, with the same lipases identified for the resolution
of both the a- and b-substituted 3-aryl alkanoic acids.
2.4. Hydrolase-catalysed kinetic resolution to provide
enantioenriched 2-benzyl-3,3-dimethylbutanoic acid 1c
4. Experimental
4.1. General
The next substrate investigated was the
ester 2c. Given the decrease in efficiency and enantioselectivity for
the -ethyl substituted (R)-2-benzylbutanoic acid (R)-1b relative
to the -methyl substituted (S)-2-methyl-3-phenylpropanoic acid
a-tert-butyl substituted
a
Solvents were distilled prior to use as follows: dichloromethane
was distilled from phosphorus pentoxide. Ethyl acetate was dis-
tilled from potassium carbonate and hexane was distilled prior to
use. Tetrahydrofuran (THF) was distilled from sodium and benzo-
phenone. Diisopropylamine was distilled from calcium hydride.
The organic phases were dried over anhydrous magnesium sulfate.
Infrared spectra were recorded as thin films on sodium chloride
plates on a Perkin Elmer Paragon 1000 FT-IR spectrometer. NMR
spectra were recorded on a 300 MHz or 400 MHz NMR spectrome-
ter. All spectra were recorded at room temperature (ꢂ20 °C) in
deuterated chloroform (CDCl3), unless otherwise stated, using tet-
a
(S)-1a, challenges in efficiency were anticipated. In the screening
assays, none of the lipases, including Candida antarctica lipase B,
successfully catalysed the hydrolysis of 2c to any extent.§ We have
previously demonstrated the ability of Candida antarctica lipase B to
resolve sterically demanding
a- and b-substituted substrates, thus
proving to be the lipase of choice for the mediated resolution of
(R)-1b, (R)-1e and (S)-1f.
The presence of the tert-butyl substituent at the
a-position
dramatically reduced the efficiency of the enzymatic hydrolysis.
Under identical reaction conditions, the extent of the resolution
of the b-substituted derivative, 4,4-dimethyl-3-phenylpentanoic
acid 1f was also extremely limited and only by increasing the
reaction temperature and extending the incubation period,
was the isolation of enantiopure samples of acid (S)-1f
ramethylsilane (TMS) as an internal standard. 1H–1H and 1H–13
C
correlations were used to confirm the NMR peak assignments. All
spectroscopic details for compounds previously made were in
agreement with those previously reported. High resolution mass
spectra (HRMS) were recorded on a Waters LCT Premier Time of
Flight spectrometer in electrospray ionisation (ESI) mode using
achieved, albeit with
a
low extent of biotransformation
(Scheme 6).7
O
O
O
Candida antarctica B
OH
OEt
OEt
35-40 °C, 88.5 h
( )-2f
2f
(R)- (23% ee)
1f
(S)- (>98% ee)
Scheme 6. Hydrolase-mediated hydrolysis of ethyl 4,4-dimethyl-3-phenylpentanoate 2f.7
§
The following hydrolases gave no conversion Pseudomonas cepacia P1, Pseudo-
monas cepacia P2, Candida antarctica B, Candida antarctica B (immob) and Pseudomo-
nas fluorescens.

R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
1485
Figure 1. Comparison of enantiomeric ratio (E-value) versus hydrolase for C2 substituted alkanoic acids 1a and 1b.
50% water/acetonitrile containing 0.1% formic acid as eluant; sam-
ples were made up in acetonitrile. Elemental analysis was per-
formed by the Microanalysis Laboratory, National University of
Ireland, Cork, using Perkin–Elmer 240 and Exeter Analytical
CE440 elemental analysers. Enantiomeric excesses were measured
by high performance liquid chromatography (HPLC), using a
ChiralcelÒ OJ-H column (5 ꢃ 250 mm) from Daicel Chemical Indus-
tries Limited. Mobile phase, flow rate, detection wavelength and
temperature are included in the appropriate Tables 1 and 3. HPLC
analysis was performed on a Waters alliance 2690 separations
module with a PDA detector. When only one single enantiomer
could be detected, the enantiomeric excess is quoted as >98%. Opti-
cal rotations were measured on a Perkin–Elmer 141 polarimeter at
589 nm in a 10 cm cell; concentrations (c) are expressed in
JBX 12.8, C(2)H], 4.08 (2H, q, J 7.2, OCH2CH3), 7.15–7.21 [3H, m,
C(30)H, C(40)H and C(50)H, ArH], 7.25–7.28 [2H, m, C(20)H and
C(60)H, ArH].
4.2.2. Ethyl 2-benzylbutanoate 2b28
At first, n-butyllithium (1.9 M in hexanes, 27 mL, 51.45 mmol)
was added dropwise to freshly distilled diisopropylamine
(8.5 mL, 60.65 mmol) in freshly distilled tetrahydrofuran (40 mL)
at ꢁ78 °C under an atmosphere of nitrogen. Once the addition
was complete, the reaction mixture was warmed to ꢁ35 °C. Ethyl
butyrate (6.5 mL, 49.13 mmol) in tetrahydrofuran (35 mL) was
then added dropwise to the solution and once the addition was
complete, the reaction mixture was stirred for 1.5 h at ꢁ35 °C.
Benzyl bromide (6.4 mL, 53.81 mmol) was then added in one por-
tion. The reaction mixture was stirred overnight at ꢁ35 °C. The
reaction was quenched by pouring the mixture onto aqueous
hydrochloric acid (10%, 400 mL) and diethyl ether (200 mL). The
layers were separated and the aqueous layer extracted with diethyl
ether (2 ꢃ 100 mL). The combined organic layer was washed with
water (100 mL), brine (100 mL), dried, filtered and concentrated
under reduced pressure to give the crude ester 2b (10.14 g) as a
yellow oil. Purification by column chromatography on silica gel
using dichloromethane as eluent gave the pure ester 2b (5.08 g,
50%) as a clear oil; (found: C, 74.80; H, 8.73. C13H18O2 requires C,
g/100 mL. ½a 2D0
ꢀ
is the specific rotation of a compound and is ex-
pressed in units of 10ꢁ1 deg cm2 gꢁ1. All hydrolases were kindly
donated by Almac Sciences and all enzymatic reactions were per-
formed on a VWR Incubating Mini Shaker 4450.
4.2. Synthesis of ethyl esters
4.2.1. Ethyl 2-methyl-3-phenylpropanoate 2a27
Sulfuric acid (concd 95–97%, 2.1 mL, 39.4 mmol) was added to a
solution of 2-methyl-3-phenylpropanoic acid 1a (2.21 g,
13.46 mmol) in absolute ethanol (40 mL) and refluxed overnight.
Excess ethanol was evaporated off under reduced pressure. The
crude product was dissolved in dichloromethane (45 mL) and
washed with water (2 ꢃ 45 mL), a saturated aqueous solution of
sodium bicarbonate (2 ꢃ 45 mL) and brine (50 mL), dried, filtered
and concentrated under reduced pressure to give the crude ester
2a (1.67 g) as a clear oil. Purification by column chromatography
on silica gel using hexane/ethyl acetate 60/40 as eluent gave the
75.69; H, 8.80%); v
max/cmꢁ1 (film) 2966 (CH), 1732 (CO), 1605,
1496, 1456 (Ar), 1163 (C–O); dH (300 MHz) 0.92 [3H, t, J 7.4,
C(4)H3], 1.15 (3H, t, J 7.2, OCH2CH3), 1.44–1.77 [2H, m, C(3)H2],
2.53–2.62 [1H, m, X of ABX, C(2)H], 2.74 [1H, dd, A of ABX, JAB
13.5, JAX 6.6, one of CH2Ph], 2.93 [1H, dd, B of ABX, JAB 13.5, JBX
8.4, one of CH2Ph], 4.06 (2H, q, J 7.1, OCH2CH3), 7.15–7.29 (5H,
m, ArH); dC (75.5 MHz) 11.7 [CH3, C(4)H3], 14.2 (CH3, OCH2CH3),
25.2 [CH2, C(3)H2] 38.2 (CH2, CH2Ph), 49.2 [CH, C(2)H], 60.1 (CH2,
OCH2CH3), 126.2 [CH, C(40)H, ArCH], 128.3, 128.9 [4 ꢃ CH, C(20)H,
C(60)H, C(30)H and C(50)H, ArCH], 139.6 [C, C(10), ArC], 175.5 [C,
C(1)]; HRMS (ES+): Exact mass calculated for C13H18O2 [M+H]+
207.1385. Found: 207.1388.
pure ester 2a (1.62 g, 63%) as a clear oil; v
max/cmꢁ1 (film) 2979
(CH), 1733 (CO), 1605, 1496, 1455 (Ar), 1176 (C–O); dH
(400 MHz) 1.15 [3H, d, J 6.8, C(2)CH3], 1.18 (3H, t, J 7.2, OCH2CH3),
2.63–2.76 (2H, m, AB of ABX, CH2Ph), 3.01 [1H, dd, X of ABX, JAX 6.4,

1486
R. E. Deasy et al. / Tetrahedron: Asymmetry 24 (2013) 1480–1487
4.2.3. Ethyl 2-benzyl-3,3-dimethylbutanoate 2c29
was shaken at 750 rpm at 24 °C. An aliquot of the reaction mixture
(1 mL) was withdrawn at 20 h. Following a mini work-up, chiral
HPLC analysis was conducted. Conversion was estimated by an
E-value calculator at 51%.22 The reaction mixture was filtered at
20 h through a pad of CeliteÒ and the hydrolase was washed with
water (2 ꢃ 20 mL) and ethyl acetate (10 ꢃ 10 mL). The layers were
separated and the aqueous layer was extracted with ethyl acetate
(2 ꢃ 30 mL) and then acidified with aqueous hydrochloric solution
(10%) and extracted with more (3 ꢃ 30 mL) ethyl acetate. The com-
bined organic layers were washed with brine (1 ꢃ 100 mL) dried,
filtered and concentrated under reduced pressure to produce a
light yellow oil (186.8 mg). Purification by column chromatogra-
phy on silica gel using hexane/ethyl acetate 90/10 as the eluent
Potassium carbonate (0.63 g, 4.58 mmol) was added to a solu-
tion of 2-benzyl-3,3-dimethylbutanoic acid 1c (0.94 g, 4.58 mmol)
in HPLC grade acetone (40 mL). Once the addition was complete,
the reaction mixture was stirred for 10 min before iodoethane
(1.53 g, 9.81 mmol) was added in one portion. The reaction mix-
ture was stirred at room temperature overnight, and then filtered
to remove the potassium carbonate. Acetone was evaporated
under reduced pressure and at this point further filtration was per-
formed to remove excess potassium carbonate. The crude product
was dissolved in dichloromethane (50 mL) and washed with water
(2 ꢃ 20 mL), a saturated aqueous solution of sodium bicarbonate
(2 ꢃ 20 mL), aqueous hydrochloric acid (5%, 2 ꢃ 25 mL) and brine
(30 mL). The organic extract was dried, filtered and concentrated
under reduced pressure to give a mixture of 2-benzyl-3,3-dimeth-
ylbutanoic acid 1c and ethyl 2-benzyl-3,3-dimethylbutanoate 2c
(0.67 g) as a clear oil in the ratio 13:87. Purification by column
chromatography on silica gel using hexane/ethyl acetate 90/10 as
gave the pure ester (R)-2a (63.7 mg, 27%) as
a
clear oil
½
a 2D0
ꢀ
¼ ꢁ36:4 (c 1.0, CHCl3), >98% ee, lit.24
½ ꢀ
a 2D0
¼ þ28:4 (c 1.0,
CHCl3), (S)-isomer, 82% ee and the pure acid (S)-1a (76.6 mg,
37%) as a clear oil ½a D20
ꢀ
¼ þ28:0 (c 0.82, CHCl3), 96% ee, lit.23
½
a 2D0
ꢀ
¼ þ30:2 (c 0.82, CHCl3), 99% ee. 1H NMR spectra were identi-
eluent gave the pure ester 2c (0.57 g, 53%) as
a
clear oil;
cal to those for the racemic materials previously prepared.
v
max/cmꢁ1 (film) 2963 (CH), 1729 (CO), 1605, 1496, 1456 (Ar),
1152 (C–O); dH (300 MHz) 1.04 (3H, t, J 7.1, OCH2CH3), 1.05 [9H,
s, C(CH3)3], 2.45 [1H, dd, X of ABX, JAX 11.4, JBX 3.9, C(2)H], 2.79–
2.93 (2H, m, AB of ABX, CH2Ph), 3.88–4.03 (2H, sym. m, OCH2CH3),
7.13–7.27 (5H, m, ArH).
4.4.3. Synthetic scale hydrolase-mediated hydrolysis of ethyl 2-
benzylbutanoate 2b
At first, Candida antarctica lipase B (immob) (407.8 mg) was
added to ethyl 2-benzylbutanoate 2b (401.4 mg, 1.95 mmol) in a
0.1 M phosphate buffer, pH 7 (20 mL). The reaction mixture was
shaken at 750 rpm for 45 h at 24 °C, then the solution was filtered
through a pad of CeliteÒ and the hydrolase washed with water
(2 ꢃ 20 mL) and heptane (10 ꢃ 10 mL). The layers were separated
and the aqueous layer was extracted with heptane (3 ꢃ 30 mL).
The combined organic layers were washed with brine
(1 ꢃ 100 mL), dried, filtered and concentrated under reduced pres-
sure to produce the pure ester (S)-2b (172.1 mg, 43%) as a clear oil
4.3. Preparation of the analytically pure acid 1b by basic hydrolysis
of the corresponding ethyl ester 2b
4.3.1. 2-Benzylbutanoic acid 1b30
Aqueous sodium hydroxide (1 M, 6 mL) was added to ethyl
2-benzylbutanoate 2b (88.5 mg, 0.43 mmol). The reaction mixture
was heated at reflux while stirring overnight, then allowed cool
to room temperature and extracted with diethyl ether (2 ꢃ 5 mL).
The ether solution was discarded. The aqueous phase was acidified
to pH 1 with aqueous hydrochloric acid (10%) and then extracted
with diethyl ether (3 ꢃ 5 mL) and the combined organic extracts
were washed with brine (10 mL), dried, filtered and concentrated
under reduced pressure to give the pure acid 1b (50.3 mg, 66%) as
½
a 2D0
ꢀ
¼ þ6:8 (c 1.0, CH2Cl2), 26% ee. The aqueous layer was acidified
with aqueous hydrochloric solution (10%) and extracted with
(3 ꢃ 30 mL) ethyl acetate. The combined organic layers were
washed with brine (1 ꢃ 100 mL), dried, filtered and concentrated
under reduced pressure to produce the pure acid (R)-1b (66.2 mg,
19%) as a clear oil ½a D20
ꢀ
¼ ꢁ43:8 (c 1.0, CH2Cl2), 82% ee, lit.25
a light orange oil;
1496, 1456 (Ar); dH (300 MHz) 0.96 [3H, t,
v
max/cmꢁ1 (film) 2966 (OH), 1705 (CO), 1605,
7.5, C(4)H3],
½
a 2D0
ꢀ
¼ ꢁ40:0 (c 1.0, CH2Cl2), >99% ee. The conversion was estimated
J
by an E-value calculator at 24%.22 1H NMR spectra were identical to
1.50–1.77 [2H, m, C(3)H2], 2.57–2.66 [1H, m, X of ABX, C(2)H],
2.75 [1H, dd, A of ABX, JAB 13.8, JAX 6.9, one of CH2Ph], 2.98 [1H,
dd, B of ABX, JAB 13.5, JBX 7.8, one of CH2Ph], 7.09–7.34 (5H, m, ArH).
those for the racemic materials previously prepared.
Acknowledgements
4.4. Enzyme screening
This work was carried out with the financial support of IRCSET
and Eli Lilly. Nuala Maguire and Tom O’Mahony are acknowledged
for their technical assistance.
4.4.1. General procedure for the hydrolase-catalysed kinetic
resolution of the 3-aryl alkanoic ethyl esters 2a–c (analytical
scale)
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