Lipase catalysed kinetic resolutions of 3-aryl alkanoic acids

Article abstract of DOI:10.1016/j.tetasy.2010.12.019

Hydrolase catalysed kinetic resolutions leading to a series of 3-aryl alkanoic acids (≥94% ee) are described. Hydrolysis of the ethyl esters with a series of hydrolases was undertaken to identify biocatalysts that yield the corresponding acids with excellent enantiopurity in each case. Steric and electronic effects on the efficiency and enantioselectivity of the biocatalytic transformation were also explored.

Full text of DOI:10.1016/j.tetasy.2010.12.019

Tetrahedron: Asymmetry 22 (2011) 47–61
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Lipase catalysed kinetic resolutions of 3-aryl alkanoic acids
Rebecca E. Deasy a, Maude Brossat b, Thomas S. Moody b, Anita R. Maguire c,
⇑
a Department of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
b Biocatalysis Group, Almac Sciences, David Keir Building, Stranmillis Road, Belfast, BT9 5AG, United Kingdom
c Department of Chemistry & School of Pharmacy, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland
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 leading to a series of 3-aryl alkanoic acids (P94% ee) are
described. Hydrolysis of the ethyl esters with a series of hydrolases was undertaken to identify biocata-
lysts that yield the corresponding acids with excellent enantiopurity in each case. Steric and electronic
effects on the efficiency and enantioselectivity of the biocatalytic transformation were also explored.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 16 November 2010
Accepted 22 December 2010
Available online 2 February 2011
1. Introduction
naturally occurring d-lactone of algae origin,23 curcumene and cur-
cuphenol, biological important bisabolene sesquiterpenes24 and in
Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiqui-
tous enzymes belonging to the family of serine hydrolases and are
unequivocally, the most utilized enzymes in biocatalysis, providing
one of the most advantageous and versatile biocatalytic methods
in asymmetric synthesis.1–11 Hydrolases are excellent biocatalysts,
combining wide substrate specificity with high regio- and enanti-
oselectivity enabling the resolution of organic substrates with effi-
cient efficiency and selectivity.12–14 Further advantages are that
hydrolases do not require the use of labile co-factors, can be recy-
cled, can be used in both free or immobilised form and are effective
under mild, environmentally benign conditions and are biodegrad-
able. These attributes make these catalysts especially attractive for
the pharmaceutical and agrochemical industries, where the inter-
est in enantiomerically pure and specifically functionalized com-
pounds is growing continuously.2,4–11
Hydrolase catalysed kinetic bioresolution is widely used to pro-
vide highly enantioenriched chiral carboxylic acids, which are
valuable synthetic intermediates for the preparation of a variety
of compounds of biological interest. There have been many reports
on the successful resolution of 2-aryl or 2-aryloxy-propionic acids;
the former are non-steroidal anti-inflammatory drugs and the
latter an important class of herbicides.15–20 Successful hydrolase
catalysed resolution of alkanoic acids, with remotely located
methyl-branching has been reported,21,22 however, the literature
has revealed only limited success on the hydrolase mediated
kinetic resolution of 3-aryl alkanoic acids. Enantiomerically pure
3-aryl alkanoic acids are used as chiral synthons in the asymmetric
synthesis of antibacterial agents, such as (ꢀ)-malyngolide, a
the synthesis of amino acids b-methyl phenylalanaine25 and b-
methyl tyrosine.26 Within our own group, 3-aryl alkanoic acids
are utilised in the synthesis of diazoketone derivatives, which in
turn have been employed in Buchner cyclization reactions demon-
strating excellent diastereoselectivity.27,28 This key transformation
is currently under investigation in the efficient asymmetric synthe-
sis of the bicyclo[5.3.0]decane skeleton, characteristic of the
aucane sesquiterpenoids.
Hydrolase catalysed non-aqueous enantioselective esterifica-
tion of acids ( )-1a, ( )-1b, ( )-1c and ( )-1d, (Fig. 1) has previously
been reported, however substrate acids ( )-1a and ( )-1b were
esterified with a modest to slow rate resulting in very low E values
(E <2) and no ester was observed under any conditions for acids
( )-1c and ( )-1d.29 Traditional aqueous Burkholderia cepacia cata-
lysed ester hydrolysis has been described for the resolution of
3-phenylbutanoic acid ( )-1a (E >50), however this work has not
been expanded to include acid substrates ( )-1b, ( )-1c and ( )-
1d, encompassing more sterically hindered substituents at the ste-
reogenic centre.30
Herein, we wished to explore a wide range of hydrolases to
establish if it was possible to generate the carboxylic acid
( )-1a–i in enantiopure form through kinetic bioresolution. Acids
( )-1a–d, were selected for investigation to determine the impact
of steric effects at C3 on the efficiency of the kinetic resolution,
while substrates ( )-1e–i were designed to explore both steric
and electronic effects of substituents on the aromatic ring on the
biotransformations. In contrast to the limited reported success in
enantioselective esterification, this study focussed on enantioslec-
tive hydrolysis and indeed it was found that through an appropri-
ate choice of biocatalyst and reaction conditions, each of the
carboxylic acids could be obtained in highly enantioenriched
form.
⇑
Corresponding author. Tel.: +353 21 4901693; fax: +353 21 4901770.
E-mail address: a.maguire@ucc.ie (A.R. Maguire).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.12.019

48
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
O
O
O
O
O
OH
OH
OH
OH
R1
OH
( )-1c
( )-1d
( )-1e-i
( )-1a
( )-1b
( )-1e R1=p-CH3
( )-1f R1=m-CH3
( )-1g R1=o-CH3
( )-1h R1=p-OMe
( )-1i R1=p-F
Figure 1.
which both enantiomers of the ester and acid could be seen on a
single trace (Fig. 2). With a single injection, ready monitoring of
both the efficiency and stereoselectivity of each of the hydrolase
mediated transformations could be performed.
2. Results and discussion
2.1. Synthesis of ethyl 3-aryl alkanoates
Racemic ester ( )-3a was obtained via a simple Fischer esterifi-
cation reaction from commercial 3-phenylbutanoic acid ( )-1a
(Scheme 1). The 3-aryl alkanoic esters ( )-3b–h were synthesized
in a three step synthesis. Acids ( )-1b–d were synthesised by the
conjugate addition of an alkyl Grignard to cinnamic acid, while
acids ( )-1e–h were similarly prepared by the conjugate addition
of the appropriate aryl Grignard to crotonic acid.31 When the direct
esterification of the crude carboxylic acid was attempted, it was
found that it was simpler to obtain the ethyl esters in analytically
pure form by first transforming the carboxylic acid ( )-1b–h iso-
lated from Grignard additions directly to the analogous acid chlo-
ride, which was readily purified by vacuum distillation. Treatment
of the pure acid chloride with ethanol in the presence of triethyl-
amine led to an analytically pure ester (Scheme 1). An alternative
route via a Wadsworth–Emmons reaction was employed in the
synthesis of ethyl 3-(4-fluorophenyl)butanoate ( )-3i (Scheme 2).32
Acids 1a–e and 1h have been previously reported in the literature
in enantioenriched form and therefore the assignment of the abso-
lute stereochemistry for each of these compounds was made by
comparison of specific rotation data. Acids 1f–g and 1i have not
been previously reported in enantiopure form and the absolute ste-
reochemistry was determined in each case through crystallogra-
phy studies.
2.1.1. Hydrolase catalysed kinetic resolution of ( )-3-
phenylbutanoic acid ( )-1a
In total, 21 lipases and 1 esterase were screened in resolving
racemic 3-phenylbutanoic acid ( )-1a. All of the hydrolases inves-
tigated resulted in the hydrolysis of ethyl 3-phenylbutanoate
( )-3a to a certain extent and the screening results are summarised
in Table 1. Pseudomonas cepacia, Alcaligenes spp. and Pseudomonas
fluorescens, entries 6, 11 and 15, respectively, exhibited excellent
enantioselection in the hydrolysis of substrate ( )-3a. Burholderia
cepacia hydrolysis of the methyl ester of ( )-1a had previously
been reported (E >50) providing access to the acid (S)-1a with
89% ee.30 Herein, Alcaligenes spp. yielded the acid (S)-1a with excel-
lent improved enantioselectivity of 97% ee (E >200) by hydrolysis
of the corresponding ethyl ester ( )-3a. Unreacted (R)-3a was
recovered in 98% ee providing access to both enantiomeric series
in a single resolution.
From Table 1, it is evident that certain hydrolases preferentially
hydrolysed the (R)-enantiomer of substrate ( )-3a, providing
access to the complementary enantiomer (R)-1a. Candida antarctica
lipase B hydrolysis of ( )-3a had previously been reported to yield
(R)-1a, (E = 9),33 while in this work, (Table 1, entry 16) it is clear
that the (R) ester is selectively hydrolysed albeit with very low
enantioselectivity. The less common pathway involving selective
hydrolysis of the (R)-ester has been successfully extended in this
With racemic samples of both the esters and acids in hand, chi-
ral HPLC conditions were developed for each ester hydrolysis in
O
OH
( )-1a
(v)
O
OH
OH
(i)
R2
O
R2
O
R2
O
(iii)
(iv)
OH
Cl
OEt
R1
R1
R1
MgBr
O
R1
(ii)
( )-1b R1=H, R2=CH2CH3
( )-1c R1=H, R2=CH(CH3)2
( )-1d R1=H, R2=C(CH3)3
( )-1e R1=p-CH3, R2=CH3
( )-1f R1=m-CH3, R2=CH3
( )-1g R1=o-CH3, R2=CH3
( )-1h R1=p-OMe, R2=CH3
( )-2b R1=H, R2=CH2CH3
( )-2c R1=H, R2=CH(CH3)2
( )-2d R1=H, R2=C(CH3)3
( )-2e R1=p-CH3, R2=CH3
( )-2f R1=m-CH3, R2=CH3
( )-2g R1=o-CH3, R2=CH3
( )-2h R1=p-OMe, R2=CH3
( )-3a R1=H, R2=CH3
( )-3b R1=H, R2=CH2CH3
( )-3c R1=H, R2=CH(CH3)2
( )-3d R1=H, R2=C(CH3)3
( )-3e R1=p-CH3, R2=CH3
( )-3f R1=m-CH3, R2=CH3
( )-3g R1=o-CH3, R2=CH3
( )-3h R1=p-OMe, R2=CH3
Scheme 1. Synthesis of ethyl 3-aryl alkanoate ( )-3a–h. Reagents: (i) R2MgX, Et2O, ( )-1b–d; (ii) Et2O, ( )-1e–h; (iii) SOCl2; (iv) Et3N, EtOH, CH2Cl2; (v) EtOH, cat. H2SO4,
( )-1a.

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
49
O
O
O
O
(iii)
(i)
(ii)
OH
OEt
OEt
F
F
F
F
3i
( )-
( )-1i
Scheme 2. Synthesis of ethyl 3-(4-fluorophenyl)butanoate ( )-3i and ethyl 3-(4-fluorophenyl)butanoic acid ( )-1i. Reagents: (i) (C2H5O)2P(O)CH2CO2Et, NaH, THF; (ii) H2, Pd/
C, EtOH; (iii) NaOH.32
64.716
(S)-1f
85.398
(c)
37.256
R)-3f
(b)
66.675
37.523
42.252
88.467
( )-3f
( )-1f
(a)
Figure 2. An overlay of HPLC traces of; (a) a racemic mixture of ethyl 3-(3-methylphenyl)butanoic acid ( )-1f and ethyl 3-(3-methylphenyl)butanoate ( )-3f; (b) enantiopure
ethyl 3-(3-methylphenyl)butanoate (R)-3f and (c) enantiopure 3-(3-methylphenyl)butanoic acid (S)-1f. See Table 7 for chiral HPLC conditions.
Table 1
Hydrolase-mediated hydrolysis of ( )-ethyl 3-phenylbutanaoate ( )-3a
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
65 h
( )-3a
(R)-3a
(S)-1a
Entry
Enzyme
Temperature (°C)
eea (%)
Conversionb (%)
E valueb
Ester 3a
Acid 1a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Candida cyclindracea C1
Candida cyclindracea C2
Rhizopus oryzae
Achromobacter spp.
Alcaligenes spp. 1
Pseudomonas cepacia P1
Pseudomonas stutzeri
Rhizopus spp.
Rhizopus niveus
Aspergillus niger
Alcaligenes spp. 2
Pseudomonas cepacia P2
Mucor javanicus
30
Ambient
30
30
30
11 (S)
25 (S)
—
11 (R)
27 (R)
99 (R)
14 (R)
—
—
—
98 (R)
96 (R)
—
58 (R)
59 (R)
—
90 (S)
95 (S)
94 (S)
61 (S)
—
—
—
97 (S)
75 (S)
—
16
30
4.2
4.9
—
21
50
170
4.7
—
—
—
<10c
11
22
51
19
30
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
30
Ambient
Ambient
Ambient
Ambient
Ambient
30
<10c
<10c
<10c
50
>200
26
—
56
<10c
<10c
51
Penicillium camembertii
Pseudomonas fluorescens
Candida antarctica lipase B
Mucor meihei
—
—
—
99 (R)
13 (S)
3 (S)
10 (R)
70 (S)
15 (R)
35 (R)
—
94 (S)
0 (R)
24 (R)
68 (S)
5 (R)
93 (S)
95 (S)
—
170
1.4
1.7
5.8
1.8
31
51
11
13
93
Candida antarctica lipase A
Candida antarctica lipase B (immob)
Porcine pancrease Type II
Porcine pancrease Grade II
Pig liver esterase
14
27
54
Ambient
<10c
—
a
b
c
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 95:5, flow rate 0.5 mL/min, 0 °C, k = 209.8 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3a (ees) and product acid 1a (eep).34
Estimated by chiral HPLC.

50
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
study to include the hydrolases Candida Cyclindracea and Mucor
meihei.33
reduction of enzymatic activity. Just six of the hydrolases resulted
in conversion as summarised in Table 2.
To demonstrate the practical viability of this process, Pseudomo-
nas fluorescens was selected as the most appropriate hydrolase for
preparative scale use. The conversion and enantiopurity of the es-
ters and acids were analysed utilising chiral HPLC and after 64 h,
50% conversion was achieved with 98% eeacid and 99% eeester corre-
sponding to an E value >200. Enantiopure samples of (S)-1a and
(R)-3a were isolated by chromatography in 34% and 35% yield,
respectively. Column chromatography proved more effective than
acid-base extraction for recovery of the ester and acid in this
instance.
In practise, while the use of Candida antarctica lipase B, Candida
Cyclindracea and Mucor meihei all lead selectively to (R)-1a, access
to the enantiopure (R)-1a is more effectively achieved via isolation
of enantiopure (R)-3a using Pseudomonas cepacia, Alcaligenes spp.
and Pseudomonas fluorescens, followed by saponification.
Significantly the biocatalysts which had yielded the most effec-
tive kinetic bioresolution with ( )-3a were ineffective for the enzy-
matic hydrolysis of ( )-3b. For the six biocatalysts which resulted
in ester hydrolysis, the enantioselectivities were modest at best
(Table 2). Interestingly with entries 2, 3 and 5, the poor enantiose-
lectivity is associated with a lack of discrimination of the enantio-
mers by the biocatalyst with conversions P78% in each case, while
the same biocatalysts with ( )-3a gave very limited reaction. The
immobilised Candida antarctica lipase B provided the best results
in this instance (E = 25), this is in direct contrast to the limited
reaction of ( )-3a with Candida antarctica lipase B (immob).
The direction of enantioselection in the hydrolysis of ( )-3b was
consistent with that observed in the reactions of ( )-3a with Can-
dida antarctica lipase B, Candida antarctica lipase B (immob) and
Pig liver esterase providing the (R) acid selectively. Based on the
initial promising results with Candida antarctica lipase B (immob),
the reaction conditions for the hydrolysis were varied to determine
if the outcome could be optimised.
2.1.2. Hydrolase catalysed kinetic resolution of ( )-3-
phenylpentanoic acid ( )-1b
The enzymatic hydrolysis of ( )-ethyl 3-phenylpentanoate ( )-
3b proved to be significantly less facile than with ( )-3a. Of the
16 hydrolases screened, many displayed no catalytic activity for
the hydrolysis of the substrate ethyl 3-phenylpentanoate ( )-3b.
Thus replacement of the methyl with the slightly larger ethyl moi-
ety at the stereogenic centre C3 resulted in a very significant
Temperature control in hydrolase-catalysed resolutions has
been explored due to its simplicity and reliability for enhancement
of enantioselectivity, albeit at the expense of longer reaction
times.7,35,36 Thus Candida antarctica lipase B (immob) resolution
of ( )-3b, was performed at 4 °C, (Table 2, entry 7) resulting in a
decrease in conversion even with an extended incubation period.
Table 2
Hydrolase-mediated hydrolysis of ( )-ethyl 3-phenylpentanoate ( )-3b
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer , pH 7
( )-3b
1b
(R)-
(S)-3b
Entry
Enzymea
Time (h)
Temperature (°C)
eeb (%)
Conversionc (%)
E valuec
Ester 3b
Acid 1b
1
2
3
4
5
6
7
Candida cyclindracea C2
Candida antarctica lipase B
Mucor meihei
Candida antarctica lipase A
Pig liver esterase
120
65
67
67
65
65
72
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
4
—
—
<10d
78
100
10
85
51
—
3.4
—
2.7
3.1
25
80 (S)
23 (R)
e
e
e
—
—
5 (R)
87 (S)
85 (S)
62 (S)
44 (S)
15 (R)
81 (R)
86 (R)
Candida antarctica
Lipase B (immob)
42
24
a
The following hydrolases gave no conversion Pseudomonas cepacia P2, Pseudomonas cepacia P1, Alcaligenes spp. 2, Pseudomonas fluorescens, Porcine Pancrease Type II,
Pseudomonas stutzeri, Rhizopus niveus, Candida cyclindracea C1, Aspergillus niger and Mucor javanicus.
b
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 99:1, flow rate 0.5 mL/min, 0 °C, k = 209.8 nm].
c
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3b (ees) and product acid 1b (eep).34
d
Estimated by chiral HPLC.
Reaction went to 100% completion and no enantioselectivity observed.
e
Table 3
Investigation of co-solvent effect on Candida antarctica lipase B (immob) hydrolysis of ( )-ethyl 3-phenylpentanoate ( )-3b
Entry
Co-solvent
Time (h)
Temperature (°C)
eeb (%)
Conversionc (%)
E valuec
Ester (S)-3b
Acid (R)-1b
1
2
3
4
5
6
DMSO
64
64
64
64
64
64
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
93
28
25
6
72
24
81
93
94
88
92d
57
53
23
21
6
44
30
31
36
41
16
51
4.6
Acetonitrilea
Acetonea
THF
Dioxane
TBMEa
a
b
c
HPLC grade.
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 99:1, flow rate 0.5 mL/min, 0 °C, k = 209.8 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3b (ees) and product acid 1b (eep).34
On one occasion, the enantiomeric excess isolated from dioxane of (R)-1b was 97% ee.
d

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
51
No significant increase in enantioselection was observed, therefore
this approach was not pursued further.
Candida antarctica lipase B (immob). In this instance (S)-1c was ob-
tained in 99% ee using Candida antartica lipase B; hence no further
optimisation was required.
The utilisation of organic co-solvents has been well established
to increase the enantioselectivity of hydrolase catalysed resolution
of an extensive range of compounds.10,37,38 Screening reactions
were therefore performed to assess the effect of a series of co-
solvents (at 17% v/v) on the Candida antarctica lipase B (immob)
resolution of ( )-3b (Table 3). The majority of co-solvents investi-
gated resulted in a decrease in the rate of hydrolysis, but notably,
with the exception of TBME, resulted in an equivalent or an im-
proved enantiopurity of (R)-1b. The utilisation of acetone as an
additive, (Table 3, entry 3) resulted in the recovery of (R)-1b with
94% ee and E = 41 while with dioxane (Table 3, entry 5) E = 51. Thus
the hydrolase catalysed resolution can be effective as a route to
enantioenriched (R)-1b provided the biocatalyst and reaction con-
ditions are chosen carefully. The only prior report of hydrolase cat-
alysed esterification of 1b describes very low activity and
enantioselectivity (E <2).29 Furthermore, acid (S)-1b has been re-
solved using amidase biocatalysis and again the enantiopurity
was lower (88% ee).39
Once again, the careful control of the reaction conditions and
the selection of biocatalyst led to efficient bioresolution of (S)-1c,
which is in contrast to the literature report which states that it
was not possible to resolve this acid using hydrolase catalysis.29
A solvent screen involving dioxane, acetone and TBME was con-
ducted for Candida antarctica lipase A and Candida antarctica lipase
B resolution of ( )-3c to investigate the effect on enantiomeric ex-
cess, but resulted in a significant reduction in activity and therefore
was no longer pursued.
2.1.4. Hydrolase catalysed kinetic resolution of ( )-4,4-
dimethyl-3-phenylpentanoic acid ( )-1d
The hydrolase catalysed resolution of ethyl 4,4-dimethyl-3-phe-
nylpentanoate ( )-3d was achieved using the same biocatalysts,
which catalysed the reaction of ( )-3b and ( )-3c, albeit at much
lower extent of conversion, presumably due to the increased steric
demand of the C3 substituent. However, the overall trends are very
similar for ( )-3b, ( )-3c and ( )-3d with the optimum results
being achieved with the immobilised or free Candida antarctica
Lipase B, (Fig. 3). While the extent of the reaction at room temper-
ature was extremely limited, increasing the temperature improved
the conversion, for example see entries 2 and 6, Table 5. The direc-
tion of enantioselectivities is consistent with earlier observations
for Candida antarctica lipase B (immob) and Candida antarctica li-
pase A. Interestingly the sense of enantioselection in the Pig liver
esterase hydrolase, resulting in the selective hydrolysis of the (R)-
enantiomer, is opposite to that seen in the hydrolysis of the corre-
sponding ethyl derivative ( )-3b. Thus, in the ethyl derivative ( )-
3b, Candida antarctica lipase A provided the (S)-enantiomer of the
acid selectively while Pig liver esterase provides the (R)-enantiomer
selectively, whereas the t-butyl derivative Pig liver esterase displays
the same direction of enantioselection as Candida antarctica lipase
A.
With both the free and immobilised Candida antarctica lipase B,
while the extent of the hydrolyse is limited the enantioselectivity
is excellent, with the acid (S)-1d isolated in enantiopure form.
Increasing the temperature improved the conversion, thereby
resulting in an increased enantiopurity of the unreacted ester
(R)-3d.
It is evident that once the alkyl group at the C3 stereogenic cen-
tre increases in size greater than a methyl substituent, a large de-
crease in the efficiency of the hydrolysis and thereby the kinetic
2.1.3. Hydrolase catalysed kinetic resolution of ( )-4-methyl-3-
phenylpentanoic acid ( )-1c
Due to the decrease in biocatalytic activity on increasing the C3
substituent from methyl to ethyl it was anticipated that enzymatic
hydrolysis to form (S)-1c and (S)-1d with the more sterically
demanding i-propyl and t-butyl substituents would prove extre-
mely challenging. Of the 19 hydrolases screened, many displayed
no hydrolytic activity towards ( )-3c and hydrolysis failed to occur
even at elevated temperature and extended reaction periods. Sig-
nificantly, the hydrolases that were identified to hydrolyse ( )-3b
were found to hydrolyse ( )-3c as depicted in Table 4, thus con-
firming that these biocatalysts can accommodate the increased
steric demand in the C3 region of the enzyme pocket.
Interestingly the extent of reaction in entries 1, 2 and 4, Table 4,
is decreased somewhat relatively to those seen with ( )-3b in
Table 2, resulting in improved enantiopurities of the recovered acid
(S)-1c. Thus discrimination between the phenyl and i-propyl
groups in the active site of the enzymes is improved compared to
that seen in ( )-3b where discrimination between the ethyl and
phenyl substituents is quite poor. While the (R) and (S) labels in
the acid (S)-1c are switched relative to acids (R)-1a and (R)-1b
the sense of enantioselection is identical in hydrolysis of the ethyl
and i-propyl esters ( )-3b and ( )-3c with the (S)-enantiomer iso-
lated using enzymes Candida antarctica lipase B, Mucor meihei and
Table 4
Hydrolase-mediated hydrolysis of ( )-ethyl 4-methyl-3-phenylpentanoate ( )-3c
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
65 h
( )-3c
(R)-3c
(S)-1c
Entry
Enzymea
eeb (%)
Conversionc (%)
E valuec
Ester 3c
Acid 1c
1
2
3
4
5
Candida antarctica lipase B
Mucor meihei
Candida antarctica lipase A
Candida antarctica lipase B (immob)
Pig liver esterase
12 (R)
61 (R)
10 (S)
33 (R)
99 (S)
23 (S)
64 (R)
97 (S)
11
73
14
25
100
>200
2.7
5
90
d
d
d
—
—
—
a
The following hydrolases gave no conversion Pseudomonas cepacia P2, Pseudomonas cepacia P1, Alcaligenes spp. 1, Penicillium camembertii, Pseudomonas fluorescens, Porcine
Pancrease Type II, Candida cyclindracea C2, Rhizopus spp., Pseudomonas stutzeri, Rhizopus niveus, Candida cyclindracea C1, Aspergillus niger, Alcaligenes spp. 2 and Mucor
javanicus.
b
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 98:2, flow rate 0.5 mL/min, 0 °C, k = 209.8 nm].
c
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3c (ees) and product acid 1c (eep).34
d
Reaction went to 100% completion, no enantioselectivity observed.

52
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
Figure 3. Graph of enantiomeric ratio (E) versus hydrolase; kinetic bioresolutions for 1a–d performed under standard aqueous conditions.
Table 5
Hydrolase-mediated hydrolysis of ( )-ethyl 4,4-dimethyl-3-phenylpentanoate ( )-3d at variable temperature
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
( )-3d
1d
(S)-
(R)-3d
Entry
Enzymea
Temperature °C
eee (%)
Conversionf (%)
E valuef
Ester 3d
Acid 1d
1
2
3
4
5
6
7
8
Candida antarctica lipase B
Candida antarctica lipase A
Ambientb
35–40 °Cc
Ambientb
35–40 °Cd
Ambientb
35–40 °Cc
Ambientb
35–40 °Cc
2 (R)
23 (R)
3 (S)
7 (S)
1 (R)
P99h (S)
P99h (S)
73 (R)
2
19
4
8
1
23
48
100
>200
>200
6.6
81 (R)
10
Candida antarctica lipase B (immob)
P99h (S)
98 (S)
>200
132
2.7
30 (R)
32 (S)
Pig liver esterase
34 (R)
g
g
g
—
—
—
a
The following hydrolases gave no conversion; Pseudomonas cepacia P1, Rhizopus niveus, Pseudomonas fluorescens, Candida cyclindracea C1, Pseudomonas cepacia P2 and
Porcine Pancrease Type II.
b
Time for ester hydrolysis was 66 h.
Time for ester hydrolysis was 64.5 h at 35 °C temperature increased to 40 °C for the final 24 h.
Time for ester hydrolysis was 72 h at 35 °C temperature increased to 40 °C for the final 24 h.
c
d
e
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 96:4, flow rate 0.25 mL/min, 20 °C, k = 209.8 nm].
f
Conversion and the enantiomeric ratio E was calculated from enantiomeric excess of substrate ester 3d (ees) and product acid 1d (eep).34
g
Reaction went to 100% completion, no enantioselectivity observed.
When the second enantiomer was not observed, the enantiomeric excess can be stated as P99% ee.
h
bioresolution with regards to the enantiopurity of the ester is ob-
served. Despite the steric hindrance within the active site, 3-aryl
alkanoic carboxylic acids 1a–d can be obtained through optimisa-
tion of the reaction conditions with excellent enantioselectivity.
Acid (S)-1a was obtained in 98% ee, through Pseudomonas fluores-
cens catalysed hydrolysis of ( )-3a, while acids (R)-1b, (S)-1c and
(S)-1d were obtained in P94% ee via immobilised or free Candida
antarctica lipase B catalysed kinetic bioresolution (Fig. 3).
It is noteworthy that Candida antarctica lipase A provides a via-
ble route to the complementary enantiomers (S)-1b and (R)-1c–d
and overcomes the limitation of the modest enantiomeric excess
of the esters achieved in the resolutions using the free and immo-
bilised Candida antarctica lipase B. In addition, Candida antarctica li-
pase A has a unique ability to accept very bulky, highly sterically
hindered substrates and this correlated with the observations
herein whereby the enantiopurity of the acid obtained via Candida
antartica lipase A catalysed resolution improved as the size of the
alkyl substituent at C3 increased, the highest enantiopurity ob-
tained being of (R)-1d at 81% ee (Table 5, entry 4).40,41
2.1.5. Hydrolase catalysed kinetic resolution of substituted
phenyl butanoic acids ( )-1e–i
A series of substituted phenyl butanoic acids were selected to
study the impact of the substituents on the aryl ring on the effi-
ciency of the kinetic bioresolution process. The substrates selected
were ortho-, meta- and para-tolylbutanoic acid and para-methoxy,
para-fluoro-phenyl butanoic acids in order to study the electronic
effect in addition to the impact of the position of substitution
(Fig. 4).
The results of the enzymatic screens are summarised in
Tables 6-10. In each case, effective kinetic bioresolution was achi-
eved with Pseudomonas cepacia P1, Pseudomonas cepacia P2 and

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
53
O
the recovered acids. The use of the Candida cyclindracea biocatalyst
with the substituted substrates was also explored; while Candida
cyclindracea had resulted in some hydrolysis with the parent com-
pound ( )-3a, very little conversion was seen with the substituted
derivatives (Fig. 5).
OH
R1
R1 = o, m, p-Me
R
R
1 = p-OMe
1 = p-F
The use of Candida antarctica Lipase B (immob) with the parent
substrate ( )-1a provided access to the opposite enantiomeric ser-
ies (Table 1, entry 19), albeit with a very high extent of reaction. In
general similar reaction patterns were seen with the substituted
substrates resulting in recovery of the (S)-esters 3e–f and 3h and
the (R)-acids 1e–f and 1h. Notably, with the p-Me substrate
( )-3e the extent of reaction was less, resulting in a decrease in
the enantiopurity of the recovered ester (S)-3e, while with the
para-F substrate both enantiomers are completely indiscriminately
hydrolysed. The sense of enantioselection in Candida antarctica li-
pase B (immob) resolution of the o-Me substrate ( )-3g was the
same as that seen with the Pseudomonas biocatalysts. This biocat-
alyst was the one that was able to accommodate the increased ste-
ric demand at the C3 position. The switch in enantioselection must
be due to combined steric effects of the ortho-Me and 3-Me substit-
uents, possibly via conformational changes, (Fig. 6).
Figure 4.
Pseudomonas fluorescens resulting in the successful hydrolysis of
the S enantiomer with very similar outcomes to those seen with
3-phenylbutanoic acid (S)-1a, thus indicating that the aryl substi-
tuent had little impact on the enzymatic hydrolysis.
In all cases highly enantioenriched samples of the (3S)-acids
and the (3R)-esters are readily obtained using the Pseudomonas
biocatalysts, see Figure 5. 3-(4-Methylphenyl)butanoic acid
( )-1e had previously been resolved utilising Pseudomonas cepacia
immobilized on ceramic particles to yield (S)-1e in 99% ee.24 The
results obtained utilising the free hydrolase (Table 6, entry 1) cor-
relate strongly. The only significant effect of the substituent seen in
this series of substituted phenyl butanoic acids was with the para-F
substrate ( )-3i where the conversion is increased relative to the
other substrates resulting in a slight decrease in enantiopurity of
As summarised in Table 11, nine of the hydrolyses were scaled up
to synthetic batches leading to isolations of acids 1a–i and esters
3a–i in excellent enantiopurity in most cases. It is noteworthy that
Table 6
Hydrolase-mediated hydrolysis of ( )-ethyl 3-(4-methylphenyl)butanoate ( )-3e
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
64 h
3e
3e
(R)-
( )-
(S)-1e
eea (%)
Entry
Enzyme
Temperature (°C)
Conversionb (%)
E valueb
Ester 3e
Acid 1e
1
2
3
4
5
6
Pseudomonas cepacia P1
Pseudomonas cepacia P2
Pseudomonas fluorescens
Candida cyclindracea
Candida antarctica lipase A
Candida antarctica lipase B (immob)
30
30
30
30
30
30
98 (R)
99 (R)
99 (S)
96 (S)
95 (S)
—
68 (S)
5 (R)
50
51
51
0
7
55
>200
>200
>200
—
5.5
1.2
P99d (R)
c
c
c
—
5 (R)
6 (S)
a
b
c
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 99.5:0.5, flow rate 0.5 mL/min, 0 °C, k = 211 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3e (ees) and product acid 1e (eep).34
Reaction failed to proceed; no enantioselectivity observed.
d
When the second enantiomer was not observed, the enantiomeric excess could be stated as P99% ee.
Table 7
Hydrolase-mediated hydrolysis of ( )-ethyl 3-(3-methylphenyl)butanoate ( )-3f
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
( )-3f
1f
(S)-
(R)-3f
Entry
Enzyme
Temperature (°C)
eea,b (%)
Conversionc (%)
E valuec
Ester 3f
Acid 1f
1
2
3
4
5
Pseudomonas cepacia P1
Pseudomonas cepacia P2
Pseudomonas fluorescens
Candida cyclindracea
30
30
30
30
30
88 (R)
P99e (R)
96 (R)
96 (S)
76 (S)
97 (S)
—
7 (R)
48
57
50
0
143
52
>200
d
d
d
—
—
Candida antarctica Lipase B (immob)
P99e (S)
93
4.7
a
b
c
Time for ester hydrolysis was 65 h with the exception of Candida cyclindracea catalysed hydrolysis which was 64 h.
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 98:2, flow rate 0.25 mL/min, 0 °C, k = 209.8 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3f (ees) and product acid 1f (eep).34
Reaction failed to proceed, no enantioselectivity observed.
d
e
When the second enantiomer was not observed, the enantiomeric excess could be stated as P99% ee.

54
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
Table 8
Hydrolase-mediated hydrolysis of ( )-ethyl 3-(2-methylphenyl)butanoate ( )-3g
O
O
O
Hydrolase
OEt
OH
OEt
0.1 M phosphate buffer, pH 7
( )-3g
1g
(S)-
(R)-3g
Entry
Enzyme source
Temperature (°C)
eea,b (%)
Conversionc (%)
E valuec
Ester 3g
Acid 1g
1
2
3
4
5
Pseudomonas cepacia P1
Pseudomonas cepacia P2
Pseudomonas fluorescens
Candida cyclindracea
30
30
30
30
30
P99e (R)
P99e (R)
P99e (R)
—
90 (R)
99 (S)
80 (S)
50
56
50
0
>200
65
P99e (S)
>200
d
d
d
—
—
Candida antarctica Lipase B (immob)
46 (S)
66
7.7
a
b
c
Time for ester hydrolysis was 67 h with the exception of Pseudomonas fluorescens catalysed hydrolysis which was 64 h.
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 94:6, flow rate 0.25 mL/min, 0 °C, k = 209.8 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3g (ees) and product acid 1g (eep).34
Reaction failed to proceed, no enantioselectivity observed.
d
e
When the second enantiomer was not observed, the enantiomeric excess could be stated as P99% ee.
Table 9
Hydrolase-mediated hydrolysis of ( )-ethyl 3-(4-methoxyphenyl)butanoate ( )-3h
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
68 h
MeO
MeO
MeO
3h
( )-
3h
(R)-
Conversionb (%)
(S)-1h
eea (%)
Entry
Enzyme
Temperature (°C)
E valueb
Ester 3h
Acid 1h
1
2
3
4
5
6
Pseudomonas cepacia P1
Pseudomonas cepacia P2
Pseudomonas fluorescens
Candida Antarctica Lipase A
Candida cyclindracea
Candida antarctica Lipase B (immob)
30
30
30
30
30
30
98 (R)
99 (R)
P99d (R)
4 (R)
—
66 (S)
86 (S)
88 (S)
97 (S)
48 (S)
—
53
53
51
60
81
>200
3
—
1.9
8
<10c
90
7 (R)
a
b
c
Determined by chiral HPLC analysis [Daicel Chiralcel OJ-H, hexane/i-PrOH (3% trifluoroacetic acid) = 82:18, flow rate 0.25 mL/min, 0 °C, k = 216.9 nm].
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3h (ees) and product acid 1h (eep).34
Estimated by chiral HPLC.
d
When the second enantiomer was not observed, the enantiomeric excess could be stated as P99% ee.
Table 10
Hydrolase-mediated hydrolysis of ( )-ethyl 3-(4-fluorophenyl)butanoate ( )-3i
O
O
O
Hydrolase
OH
OEt
OEt
0.1 M phosphate buffer, pH 7
64 h
F
F
F
( )-3i
1i
(R)-3i
(S)-
Entry
Enzyme
Temperature (°C)
eea (%)
Conversionb (%)
E valueb
Ester 3i
Acid 1i
1
2
3
4
5
Pseudomonas cepacia P1
Pseudomonas cepacia P2
Pseudomonas fluorescens
Candida cyclindracea
30
30
30
30
30
P99d (R)
P99d (R)
P99d (R)
3 (S)
84 (S)
69 (S)
94 (S)
25 (R)
54
84
62
11
100
59
27
170
1.7
c
c
c
Candida antarctica Lipase B (immob)
—
—
—
a
Determined by chiral HPLC analysis [Daicel Chiralcel AS-H column, Step gradient: 0 °C, k = 256 nm, hexane/i-PrOH (3% trifluoroacetic acid), 0–30 min; 99.7:0.3, flow rate
1 mL/min. 31 min; 94:6, flow rate 0.25 mL/min].
b
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3i (ees) and product acid 1i (eep).34
Reaction went to 100% completion, no enantioselectivity observed.
When the second enantiomer is not observed enantiomeric excess is stated as P99% ee.
c
d
on scale-up, the efficiencies and selectivities mirrored quite closely
the outcomes seen in the analytical scale reactions summarised in
Tables 1–10. Notably with the para-F series, acid (S)-1i was recov-
ered in excellent enantiopurity in contrast to the small scale reac-
tion (Table 10, entry 3). With the 3-aryl butanoic acids, the
products of the biocatalysis were readily isolated by extraction with
ethyl acetate followed by chromatographic separation of the acids
and esters. With the bulkier 3-alkyl substituents, chromatography

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
55
Figure 5. Graph of enantiomeric ratio (E) versus hydrolase—kinetic bioresolutions for 1a and 1e–i performed under standard aqueous conditions.
apparent upon resolving acids ( )-1a–d that a large reduction in
reaction rate and enantioselectivity was observed once the moiety
at the C3 stereogenic centre increased in size greater than a methyl.
Despite this, the highest obtained enantiopurities of the hydrolase
catalysed bioresolutions of 3-aryl alkanoic acids ( )-1a–d are
reported through optimisation of reaction conditions and a viable
route to both enantiomers has been identified. Furthermore,
substituents on the phenyl ring, acids ( )-1e–i were determined
to have limited effect on the excellent enantioselectivities attain-
able indicating that these hydrolases can tolerate increased steric
demand in the aryl group more readily than in the 3-alkyl group.
Significantly, nine of these experiments were conducted on a
synthetic scale leading to the isolation of acids 1a–i and esters
3a–i with excellent enantiopurity being achieved in most cases.
O
OH
Figure 6.
was less effective. In practice the ester was recovered first by hep-
tane extraction of the biotransformation mixture, then subsequent
acidification and extraction with ethyl acetate provided the acids
(R)-1b and (S)-1c–d. Each of the products were characterised by
1H NMR and chiral HPLC.
3. Conclusion
4. Experimental
Herein a series of 3-aryl alkanoic acids ( )-1a–i were success-
fully resolved with enantiopurity P94% ee via hydrolase catalysed
kinetic hydrolysis of the corresponding ethyl esters. It was
All solvents were distilled prior to use as follows: dichlorometh-
ane was distilled from phosphorus pentoxide and ethyl acetate
Table 11
Synthetic scales of hydrolase-mediated hydrolysis of ( )-3a–i
R2
*
O
R2
O
R2
*
O
Hydrolase
OH
eea (%)
OEt
OEt
R1
R1
R1
0.1 M phosphate buffer, pH 7
Entry
Ester substrate
R1
Enzyme
Conversionb (%)
E valueb
R2
Ester
Acid
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
CH3
CH2CH3
CH(CH3)2
C(CH3)3
CH3
CH3
CH3
CH3
CH3
Pseudomonas fluorescens
99 (R)-3a
65 (S)-3b
26 (R)-3c
12 (R)-3d
97 (R)-3e
94 (R)-3f
98 (R)-3g
99 (R)-3h
P99d (R)-3i
98 (S)-1a
90 (R)-1b
98 (S)-1c
50
42
21
11
49
49
50
51
51
>200
37
127
>200
>200
>200
>200
>200
>200
Candida antarctica lipase B (immob)c
Candida antarctica lipase B (immob)
Candida antarctica lipase B (immob)
Pseudomonas cepacia P1
99 (S)-1d
p-CH3
m-CH3
o-CH3
p-OCH3
p-F
P99d (S)-1e
P99d (S)-1f
P99d (S)-1g
97 (S)-1h
Pseudomonas fluorescens
Pseudomonas fluorescens
Pseudomonas fluorescens
Pseudomonas fluorescens
97 (S)-3i
a
b
c
For chiral HPLC conditions see details on relevant Tables 1–10.
Conversion and the enantiomeric ratio E was calculated from the enantiomeric excess of substrate ester 3a–i (ees) and product acid 1a–i (eep).34
17% v/v dioxane co-solvent was added, see Section 4 for further information.
d
When the second enantiomer was not observed, the enantiomeric excess could be stated as P99% ee.

56
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
was distilled from potassium carbonate. Ethanol was distilled from
magnesium in the presence of iodine and stored over 3 Å molecular
sieves. Hexane was distilled prior to use. Tetrahydrofuran was dis-
tilled from sodium and benzophenone. Molecular sieves were acti-
vated by heating at 150 °C overnight. Organic phases were dried
using anhydrous magnesium sulphate. Infrared spectra were re-
corded as thin films on sodium chloride plates for oils or as potas-
sium bromide (KBr) discs for solids on a Perkin–Elmer Paragon
1000 FT-IR spectrometer.
portionwise while stirring at 0 °C, then the reaction mixture
was heated at reflux for 3 h. The reaction mixture was subse-
quently cooled to room temperature and carefully poured onto
aqueous hydrochloric acid (10%, ꢁ100 mL) and ice (ꢁ65 g). The
layers were separated and the aqueous layer was washed with
diethyl ether (3 ꢃ 50 mL). The combined organic layer was
washed with hydrochloric acid (10%, 50 mL), water (50 mL), brine
(50 mL), dried, filtered and concentrated under reduced pressure
to give the crude acid ( )-1b (5.54 g, 92%) as an orange oil which
1H (300 MHz) and 13C (75.5 MHz) NMR spectra were recorded
on a Bruker Avance 300 MHz NMR spectrometer. 1H (400 MHz)
NMR spectra were recorded on a Bruker Avance 400 MHz NMR
spectrometer. All spectra were recorded at room temperature
(ꢁ20 °C) in deuterated chloroform (CDCl3) unless otherwise sta-
ted using tetramethylsilane (TMS) as an internal standard. Chem-
ical shifts (dH & dC) are reported in parts per million (ppm)
relative to TMS and coupling constants are expressed in Hertz
(Hz).
was used without further purification. m
max/cmꢀ1 (film) 2965
(OH), 1708 (CO), 1603, 1495, 1454; dH (400 MHz) 0.79 [3H, t, J
7.4, C(5)H3], 1.46–1.83 [2H, m, C(4)H2], 2.60 [1H, dd, A of ABX,
JAB 15.6, JAX 7.9, one of C(2)H2], 2.68 [1H, dd, B of ABX, JAB 15.6,
JBX 7.1, one of C(2)H2], 2.89–3.08 [1H, m, X of ABX, C(3)H],
7.09–7.38 (5H, m, ArH).
4.2. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
Low resolution mass spectra were recorded on a Waters Quattro
Micro triple quadrupole spectrometer in electrospray ionization
(ESI) mode using 50% water/acetonitrile containing 0.1% formic
acid as eluant; samples were made up in acetonitrile. High resolu-
tion mass spectra (HRMS) were recorded on a Waters LCT premier
Time of Flight spectrometer in electrospray ionization (ESI) mode
using 50% water/acetonitrile containing 0.1% formic acid as eluant;
samples were made up in acetonitrile.
Elemental analysis was performed by the Microanalysis Labora-
tory, National University of Ireland, Cork, using Perkin–Elmer 240
and Exeter Analytical CE440 elemental analysers. Melting points
were carried out on a uni-melt Thomas Hoover Capillary melting
point apparatus and are uncorrected. Wet flash chromatography
was performed using Kieselgel Silica Gel 60, 0.040–0.063 mm
(Merck). Thin layer chromatography (TLC) was carried out on pre-
coated silica gel plates (Merck 60 PF254). Visualisation was
achieved by UV (254 nm) light detection and bromocresol green
staining.
Aqueous potassium hydroxide (20%, 35 ml) was added to a sam-
ple of 3-phenylpentanoyl chloride ( )-2b (0.5 g, 2.6 mmol) under
nitrogen. The reaction mixture was heated at reflux and stirred
at reflux overnight. The reaction mixture was then cooled to room
temperature and acidified to pH 1 with aqueous hydrochloric acid
(10%), which resulted in a white precipitate. The suspension was
then extracted with dichloromethane (3 ꢃ 50 ml), and the com-
bined organic layers where washed with brine (100 ml), dried, fil-
tered and concentrated under reduced pressure to give the acid
( )-1b (0.428 g, 92%) as a cream solid, mp 59–62 °C (Lit.,31
60–61 °C) and with spectroscopic characteristics identical to those
described above.
4.2.1. ( )-4-Methyl-3-phenylpentanoic acid ( )-1c28,42
This was prepared following the procedure described for ( )-1b
from isopropyl magnesium bromide [freshly prepared from mag-
nesium (2.70 g, 111 mmol) and iodine (catalytic amount) in diethyl
ether (30 mL), and isopropyl bromide (11.25 mL, 120 mmol) in
diethyl ether (30 mL)] and cinnamic acid (5.00 g, 33.75 mmol) to
give the crude acid ( )-1c (5.38 g, 83%) as an orange oil, which
Optical rotations were measured on a Perkin–Elmer 141 polar-
imeter at 589 nm in a 10 cm cell; concentrations (c) are expressed
in g/100 mL. ½a 2D0
ꢂ
is the specific rotation of a compound and is ex-
pressed in units of 10ꢀ1 deg cm2 gꢀ1. The hydrolases used for these
biotransformations were obtained from Almac Sciences. All re-
agents are analytical grade and purchased from Sigma Aldrich
chemical company. All enzymatic reactions were performed on a
VWR Incubating Mini Shaker 4450. The enantiomeric purity of
acids 1a–h and esters 3a–h were determined by chiral HPLC anal-
ysis on a Chiralcel OJ-H column (5 ꢃ 250 mm); the enantiomeric
purity of acid 1i and ester 3i were determined on a Chiralcel
AS-H column (5 ꢃ 250 mm) purchased from Daicel Chemical
Industries, Japan. Mobile phase, flow rate, detection wavelength
and temperature are stated in the appropriate Table 1–10. HPLC
analysis was performed on a Waters alliance 2690 separations
module with a PDA detector. All solvents employed were of HPLC
grade. Bulb to bulb distillations were carried out on an Aldrich Ku-
gelrohr apparatus and the oven temperature is given as the boiling
point of the substrate. ( )-3-(p-Fluorophenyl)butanoic acid ( )-1i
and ( )-ethyl 3-(p-fluorophenyl)butanoate ( )-3i were prepared
according to the procedure described by Speranza.32
was used without further purification. v
max/cmꢀ1 (film) 2963
(OH), 1709 (CO), 1602, 1495, 1454; dH (400 MHz) 0.74, 0.92
[2 ꢃ 3H, 2 ꢃ d, J 6.7, J 6.7, C(4)HCH3, C(5)H3], 1.79–1.91 [1H, m,
C(4)H], 2.60 [1H, dd, A of ABX, JAB 15.5, JAX 9.5, one of C(2)H2],
2.72–2.92 [2H, m, BX of ABX, one of C(2)H2, C(3)H], 7.10–7.28
(5H, m, ArH).
4.3. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 50 mL) and 4-methyl-3-
phenylpentanoyl chloride ( )-2c (1.00 g, 4.75 mmol) to give the
acid ( )-1c (900 mg, 99%) as a cream, white solid, mp 48–50 °C
(Lit.,28 46–48 °C) and with spectroscopic characteristics identical
to those described above.
4.3.1. ( )-4,4-Dimethyl-3-phenylpentanoic acid ( )-1d28,31
This was prepared following the procedure described for ( )-1b,
from t-butyl magnesium chloride (2 M in diethyl ether, 106 mL,
212 mmol) and cinnamic acid (7.85 g, 52.98 mmol) to give the
crude acid ( )-1d (8.63 g, 79%) as a yellow solid, which was used
4.1. Synthesis of carboxylic acids
4.1.1. ( )-3-Phenylpentanoic acid ( )-1b28,31,42
Ethyl magnesium bromide was freshly prepared from magne-
sium (2.85 g, 117 mmol) and iodine (catalytic amount) in diethyl
ether (30 mL) and ethyl bromide (8.7 mL, 117 mmol) in diethyl
ether (30 mL) at 0 °C under nitrogen and the mixture was stirred
for 0.5 h at 0 °C. Cinnamic acid (5.00 g, 33.75 mmol) was added
without further purification. m
max/cmꢀ1 (KBr) 2955 (OH), 1726
(CO), 1638, 1453; dH (400 MHz) 0.87 [9H, s, C(CH3)3], 2.73 [1H,
dd, A of ABX, JAB 15.8, JAX 10.8, one of C(2)H2], 2.81 [1H, dd, B of
ABX, JAB 15.8, JBX 4.5, one of C(2)H2], 2.93 [1H, dd, X of ABX, JAX
10.8, JBX 4.5, C(3)H], 7.07–7.33 (5H, m, ArH).

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
57
4.4. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
magnesium (4.22 g, 174 mmol) and iodine (catalytic amount) in
diethyl ether (40 mL), and 2-bromotoluene (21 mL, 175 mmol) in
diethyl ether (40 mL)] and crotonic acid (5.00 g, 58 mmol) to give
the crude acid ( )-1g (8.21 g, 79%) as an orange oil, which was used
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 8 mL) and 4,4-dimethyl-
3-phenylpentanoyl chloride ( )-2d (214 mg, 0.95 mmol) to give
the acid ( )-1d (153 mg, 78%) as a white solid, mp 108–110 °C
(Lit.,28 114–116 °C) and with spectroscopic characteristics identical
to those described above.
without further purification.
v
max/cmꢀ1 (film) 2973 (OH), 1712
(CO), 1605, 1492, 1460; dH (400 MHz) 1.27 [3H, d, J 6.9, C(4)H3],
2.36 [3H, s, C(20)CH3], 2.55 [1H, dd, A of ABX, JAB 15.7, JAX 8.6,
one of C(2)H2], 2.67 [1H, dd, B of ABX, JAB 15.6, JBX 6.3, one of
C(2)H2], 3.45–3.58 [1H, m, X of ABX, C(3)H], 7.06–7.27 (4H, m,
ArH).
4.4.1. ( )-3-(4-Methylphenyl)butanoic acid ( )-1e42
This was prepared from p-tolyl magnesium bromide [freshly
prepared from magnesium (10 g, 411 mmol) and iodine (catalytic
amount) in diethyl ether (80 mL), and 4-bromotoluene (51.6 mL,
419 mmol) in diethyl ether (60 mL)] and crotonic acid (12.00 g,
139 mmol). The reaction mixture, containing product ( )-1e and
the Wurtz coupling product, was acidified to pH 2 and the aqueous
layer washed with diethyl ether (2 ꢃ 100 mL). The combined
diethyl ether extracts were washed with sodium hydroxide (20%,
2 ꢃ 100 mL) and the aqueous layer was acidified to pH 1 with conc.
hydrochloric acid and extracted with diethyl ether (3 ꢃ 100 mL).
The organic layer was dried, filtered and concentrated under re-
duced pressure to give the crude acid ( )-1e as a viscous yellow
oil (21.19 g, 86%) which was used without further purification.
4.7. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 100 mL) and 3-(2-meth-
ylphenyl)butanoyl chloride ( )-2g (3.10 g, 15.75 mmol) to give the
acid ( )-1g (2.20 g, 78%) as a yellow solid, mp 46–48 °C (Lit.,45 46–
47 °C) and with spectroscopic characteristics identical to those de-
scribed above.
4.7.1. ( )-2-(4-Methoxyphenyl)butanoic acid ( )-1h46,47
This was prepared following the procedure described for ( )-1e
from p-methoxyphenyl magnesium bromide [freshly prepared
from magnesium (10.22 g, 420 mmol) and iodine (catalytic
amount) in diethyl ether (80 mL), and 4-bromoanisole (52 mL,
415 mmol) in diethyl ether (100 mL)] and crotonic acid (12.00 g,
139 mmol) to give the crude acid ( )-1h (19.17 g, 71%) as a yellow
v
max/cmꢀ1 (film) 2926 (OH), 1704 (CO), 1515, 1455, 1416; dH
(300 MHz) 1.30 [3H, d, J 7.0, C(4)H3], 2.31 [3H, s, C(40)CH3], 2.55
[1H, dd, A of ABX, JAB 15.5, JAX 8.2, one of C(2)H2], 2.65 [1H, dd, B
of ABX, JAB 15.5, JBX 6.9, one of C(2)H2], 3.18–3.30 [1H, m, X of
ABX, C(3)H], 7.11 (4H, s, ArH).
oil, which was used without further purification. v
max/cmꢀ1 (film)
2963 (OH), 1711 (CO), 1611, 1511, 1458; dH (400 MHz) 1.29 [3H,
d, J 7.0, C(4)H3], 2.54 [1H, dd, A of ABX, JAB 15.4, JAX 8.1, one of
C(2)H2], 2.62 [1H, dd, B of ABX, JAB 15.4, JBX 7.0, one of C(2)H2],
3.17–3.29 [1H, m, X of ABX, C(3)H], 3.77 (3H, s, OCH3), 6.79–6.88
[2H, m, C(30)H, C(50)H], 7.11–7.16 [2H, m, C(20)H, C(60)H].
4.5. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 8 mL) and 3-(4-methyl-
phenyl)butanoyl acid chloride ( )-2e (200 mg, 1.02 mmol) to give
the acid ( )-1e (152 mg, 83%) as a white solid, mp 91–92 °C
(Lit.,43 87–88 °C) and with spectroscopic characteristics identical
to those described above.
4.8. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 8 mL) and 3-(4-methoxy-
phenyl)butanoyl chloride ( )-2h (200 mg, 0.94 mmol) to give the
acid ( )-1h (114 mg, 62%) as a cream solid, mp 66–68 °C (Lit.,46
67–69 °C) and with spectroscopic characteristics identical to those
described above.
4.5.1. ( )-3-(3-Methylphenyl)butanoic acid ( )-1f44
This was prepared following the procedure described for ( )-1e
from 3-tolyl magnesium bromide [freshly prepared from magne-
sium (4.24 g, 174 mmol) and iodine (catalytic amount) in diethyl
ether (40 mL), and 3-bromotoluene (21 mL, 173 mmol) in diethyl
ether (40 mL)] and crotonic acid (5.00 g, 58 mmol) to give the
crude acid ( )-1f (8.55 g, 83%) as an orange oil, which was used
4.9. Synthesis of acid chlorides
without further purification.
v
max/cmꢀ1 (film) 2971 (OH), 1718
The acid chlorides were generally prepared from the crude
acids. The yield given below is the yield of acid chloride over two
steps calculated from the unsaturated precursor for the acid.
(CO), 1608, 1490, 1455; dH (300 MHz) 1.30 [3H, d, J 7.0, C(4)H3],
2.33 [3H, s, C(30)CH3], 2.55 [1H, dd, A of ABX, JAB 15.5, JAX 8.4,
one of C(2)H2], 2.66 [1H, dd, B of ABX, JAB 15.5, JBX 6.7, one of
C(2)H2], 3.14–3.31 [1H, m, X of ABX, C(3)H], 6.91–7.22 (4H, m,
ArH).
4.9.1. ( )-3-Phenylpentanoyl chloride ( )-2b28
3-Phenylpentanoic acid ( )-1b (5.01 g, 28.13 mmol) in thionyl
chloride (16 mL, 225 mmol) was heated at reflux for 3 h while stir-
ring under nitrogen. Excess thionyl chloride was evaporated under
reduced pressure to give the acid chloride ( )-2b as a brown oil.
Purification by vacuum distillation gave the acid chloride ( )-2b
(2.07 g, 31%) as a bright yellow oil; bp 72–76 °C at 0.09 mmHg
4.6. Preparation of the analytically pure acid by basic hydrolysis
of the corresponding acid chloride
This was prepared following the procedure described for ( )-1b,
from aqueous potassium hydroxide (20%, 150 mL) and 3-(3-meth-
ylphenyl)butanoyl chloride ( )-2f (4.59 g, 23.36 mmol) to give acid
( )-1f (2.87 g, 69%) as a clear oil and with spectroscopic character-
istics identical to those described above.
(Lit.,28 113–115 °C at 0.5 mmHg);
v
max/cmꢀ1 (film) 1799 (CO),
1604, 1495, 1454; dH (400 MHz) 0.80 [3H, t, J 7.4, C(5)H3], 1.58–
1.80 [2H, m, C(4)H2], 3.04–3.23 [3H, m, C(2)H2, C(3)H], 7.16–7.34
(5H, m, ArH).
4.6.1. ( )-3-(2-Methylphenyl)butanoic acid ( )-1g45
This was prepared following the procedure described for ( )-1e
from 2-tolyl magnesium bromide [freshly prepared from
4.9.2. ( )-4-Methyl-3-phenylpentanoyl chloride ( )-2c28
This was prepared following the procedure described for ( )-2b,
from crude 4-methyl-3-phenylpentanoic acid ( )-1c (5.28 g,

58
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
27.46 mmol) and thionyl chloride (16 mL, 220 mmol) to give the
crude acid chloride ( )-2c as a brown oil. Purification by distillation
gave the acid chloride ( )-2c (2.54 g, 36%) as a clear oil; bp 78–
cmꢀ1 (film) 1790 (CO), 1614, 1515, 1463; dH (400 MHz) 1.32 [3H,
d, J 7.0, C(4)H3], 3.07 [1H, dd, A of ABX, JAB 16.4, JAX 7.8, one of
C(2)H2], 3.15 [1H, dd, B of ABX, JAB 16.4, JBX 6.8, one of C(2)H2],
3.27–3.36 [1H, m, X of ABX, C(3)H], 3.78 (3H, s, OCH3), 6.84–6.87
[2H, m, C(30)H, C(50)H], 7.11–7.15 [2H, m, C(20)H, C(60)H].
80 °C at 0.15 mmHg (Lit.,28 90–94 °C at 0.08 mmHg);
m
max/cmꢀ1
(film) 1799 (CO), 1602, 1495, 1454; dH (400 MHz) 0.76, 0.97
[2 ꢃ CH3, 2 ꢃ d, J 6.7, J 6.7, C(4)HCH3, C(5)H3], 1.82–1.94 [1H, m,
C(4)H], 2.92–2.98 [1H, m, X of ABX, C(3)H], 3.18 [1H, dd, A of
ABX, JAB 16.4, JAX 9.7, one of C(2)H2], 3.34 [1H, dd, B of ABX, JAB
16.4, JAX 5.2, one of C(2)H2], 7.09–7.35 (5H, m, ArH).
4.10. Synthesis of ethyl esters
4.10.1. ( )-Ethyl 3-phenylbutanoate ( )-3a49
Sulfuric acid (1.0 mL, 18.76 mmol) was added to a solution of
3-phenylbutanoic acid ( )-1a (998 mg, 6.08 mmol) in absolute
ethanol (20 mL) and refluxed overnight. Excess ethanol was evap-
orated under reduced pressure. The crude product was dissolved in
dichloromethane (45 mL) and washed with water (2 ꢃ 45 mL), sat.
NaHCO3 (2 ꢃ 45 mL), brine (50 mL), dried, filtered and concen-
trated under reduced pressure to give the crude ester ( )-3a
(972 mg) as a clear oil. Purification by chromatography on silica
gel using hexane/ethyl acetate 60/40 as eluant gave the pure ester
4.9.3. ( )-4,4-Dimethyl-3-phenylpentanoyl chloride ( )-2d28
This was prepared following the procedure described for ( )-2b,
from crude 4,4-dimethyl-3-phenylpentanoic acid ( )-1d (8.63 g
42 mmol) and thionyl chloride (24 mL, 335 mmol) to give the
crude acid chloride ( )-2d as a brown oil. Purification by distilla-
tion gave the acid chloride ( )-2d (5.04 g, 54%) as a bright yellow
solid, bp 84–86 °C at 0.12 mmHg (Lit.,28 123–125 °C at 0.1 mmHg);
v
max/cmꢀ1 (KBr) 1793 (CO), 1601, 1494, 1453; dH (400 MHz) 0.91
[9H, s, C(CH3)3], 3.04 [1H, dd, X of ABX, J 8.9, 6.1, C(3)H], 3.26–
( )-3a (909 mg, 78%) as a clear oil; v
max/cmꢀ1 (film) 2969 (CH),
3.39 [2H, m, C(2)H2], 7.14–7.31 (5H, m, ArH).
1733 (CO), 1604, 1495, 1454 (Ar), 1174 (C–O); dH (400 MHz) 1.17
(3H, t, J 7.1, OCH2CH3), 1.30 [3H, d, J 7.0, C(4)H3], 2.53 [1H, dd, A
of ABX, JAB 15.0, JAX 8.2, one of C(2)H2], 2.61 [1H, dd, B of ABX,
JAB 15.0, JBX 7.0, one of C(2)H2], 3.23–3.32 [1H, sym. m, X of ABX,
C(3)H], 4.08 (2H, q, J 7.1, OCH2CH3), 7.17–7.34 (5H, m, ArH).
4.9.4. ( )-3-(4-Methylphenyl)butanoyl chloride ( )-2e48
This was prepared following the procedure described for ( )-2b,
from crude 3-(4-methylphenyl)butanoic acid ( )-1e (21.19 g,
119 mmol) and thionyl chloride (86 mL, 1190 mmol) to give the
crude acid chloride ( )-2e as a black oil. Purification by distillation
gave the acid chloride ( )-2e (12.09 g, 44%) as a dark orange oil, bp
4.10.2. ( )-Ethyl 3-phenylpentanoate ( )-3b50
3-Phenylpentanoyl chloride ( )-2b (1.96 g, 9.96 mmol) in
dichloromethane (10 mL) was added dropwise to a solution of tri-
ethylamine (1.7 mL, 11.9 mmol), dichloromethane (10 mL) and
distilled ethanol (1.45 mL, 24.9 mmol), at 0 °C. The reaction mix-
ture was stirred at room temperature overnight. The crude product
was dissolved in dichloromethane (30 mL) and washed with water
(2 ꢃ 50 mL), HCl (10%, 2 ꢃ 50 mL), brine (100 mL) dried, filtered
and concentrated under reduced pressure to give the crude ester
( )-3b (1.64 g) as a deep orange oil. Purification by chromatogra-
phy on silica gel using hexane/ether 97:3 as eluant gave the pure
66–68 °C at 0.2 mmHg (Lit.,48 127 °C at 0.20 mmHg); max/cmꢀ1
v
(film) 1800 (CO), 1649, 1516, 1455; dH (300 MHz) 1.32 [3H, d, J
7.0, C(4)H3], 2.32 [3H, s, C(40)CH3], 3.07 [1H, dd, A of ABX, JAB
16.4, JAX 7.9, one of C(2)H2], 3.17 [1H, dd, B of ABX, JAB 16.4, JBX
6.4, one of C(2)H2], 3.27–3.38 [1H, m, X of ABX, C(3)H], 7.04–7.21
(4H, m, ArH).
4.9.5. ( )-3-(3-Methylphenyl)butanoyl chloride ( )-2f
This was prepared following the procedure described for ( )-2b,
from crude 3-(3-methylphenyl)butanoic acid ( )-1f (8.55 g,
47.97 mmol) and thionyl chloride (28 mL, 384 mmol) to give the
crude acid chloride ( )-2f as a black oil. Purification by distillation
gave the acid chloride ( )-2f (4.59 g, 40%) as a bright yellow oil, bp
ester ( )-3b (1.49 g, 72%) as a clear oil;
v
max/cmꢀ1 (film) 2967
(CH), 1735 (CO), 1603, 1493, 1454 (Ar), 1167 (C–O); dH
(400 MHz) 0.79 [3H, t, J 7.4, C(5)H3], 1.13 (3H, t, J 7.1, OCH2CH3),
1.55–1.76 [2H, m, C(4)H2], 2.55 [1H, dd, A of ABX, JAB 15.0, JAX
8.2, one of C(2)H2], 2.63 [1H, dd, B of ABX, JAB 15.0, JBX 7.1, one of
C(2)H2], 2.96–3.04 [1H, m, X of ABX, C(3)H], 4.02 (2H, q, J 7.1,
OCH2CH3), 7.17–7.30 (5H, m, ArH).
77–80 °C at 0.2 mmHg; v
max/cmꢀ1 (film) 1800 (CO), 1608, 1491,
1456; dH (300 MHz) 1.32 [3H, d, J 6.9, C(4)H3], 2.33 [3H, s,
C(30)CH3], 3.06 [1H, dd, A of ABX, JAB 16.5, JAX 8.0, one of C(2)H2],
3.17 [1H, dd, B of ABX, JAB 16.5, JBX 6.5, one of C(2)H2], 3.25–3.36
[1H, m, X of ABX, C(3)H], 6.98–7.05 [3H, m, C(40)H, C(50)H, C(60)H,
ArH], 7.17–7.22 [1H, m, C(20)H, ArH].
4.10.3. ( )-Ethyl 4-methyl-3-phenylpentanoate ( )-3c51
This was prepared following the procedure described for ( )-3b,
from 4-methyl-3-phenylpentanoyl chloride ( )-2c (2.43 g,
11.52 mmol), triethylamine (1.9 mL, 13.82 mmol), dichlorometh-
ane (10 mL) and distilled ethanol (1.7 mL, 28.79 mmol) to yield
the crude ester ( )-3c (2.02 g) as a yellow orange oil. Purification
by chromatography on silica gel using hexane/ether 97:3 as eluant
4.9.6. ( )-3-(2-Methylphenyl)butanoyl chloride ( )-2g
This was prepared following the procedure described for ( )-2b,
from crude 3-(2-methylphenyl)butanoic acid ( )-1g (8.21 g,
46 mmol) and thionyl chloride (27 mL, 368 mmol) to give the
crude acid chloride ( )-2g as a black oil. Purification by distillation
gave the acid chloride ( )-2g (4.68 g, 41%) as a bright yellow oil, bp
gave the pure ester ( )-3c (1.6 g, 63%) as a clear oil. v
max/cmꢀ1
(film) 2961 (CH), 1736 (CO), 1602, 1494, 1453 (Ar), 1162 (C–O);
dH (400 MHz) 0.75, 0.95 [2 ꢃ 3H, 2 ꢃ d, J 6.7, J 6.7, C(4)HCH3,
C(5)H3], 1.05 (3H, t, J 7.1, OCH2CH3) 1.79–1.91 [1H, sym m,
C(4)H], 2.58 [1H, dd, A of ABX, JAB 14.9, JAX 9.9, one of C(2)H2],
2.77 [1H, dd, B of ABX, JAB 14.9, JBX 5.6, one of C(2)H2], 2.85–2.91
[1H, m, X of ABX, C(3)H], 3.95 (2H, q, J 7.1, OCH2CH3), 7.14–7.28
(5H, m, ArH).
64–66 °C at 0.09 mmHg; v
max/cmꢀ1 (film) 1801 (CO), 1605, 1492,
1459; dH (400 MHz) 1.27 [3H, d, J 7.0, C(4)H3], 2.35 [3H, s,
C(20)CH3], 3.03 [1H, dd, A of ABX, JAB 16.6, JAX 8.2, one of C(2)H2],
3.16 [1H, dd, B of ABX, JAB 16.6, JBX 6.3, one of C(2)H2], 3.54–3.63
[1H, m, X of ABX, C(3)H], 7.07–7.28 (4H, m, ArH).
4.9.7. ( )-3-(4-Methoxyphenyl)butanoyl chloride ( )-2h46
This was prepared following the procedure described for ( )-2b,
from crude 3-(4-methoxyphenyl)butanoic acid ( )-1h (19.17 g,
99 mmol), thionyl chloride (57 mL, 790 mmol) to give the crude
acid chloride ( )-2h as a black oil. Purification by distillation gave
the acid chloride ( )-2h (8.69 g, 29%) as a orange brown oil, bp
4.10.4. ( )-Ethyl 4,4-dimethyl-3-phenylpentanoate ( )-3d52
This was prepared following the procedure described for ( )-3b,
from 4,4-dimethyl-3-phenylpentanoyl chloride ( )-2d (5.04 g,
22.4 mmol), triethylamine (3.8 mL, 26.9 mmol), dichloromethane
(30 mL) and distilled ethanol (3.3 mL, 56.1 mmol) to yield the
crude ester ( )-3d (3.81 g) as a bright yellow oil. Purification by
102–110 °C at 0.35 mmHg (Lit.,46 100 °C at 0.5 mmHg); vmax
/

R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
59
chromatography on silica gel using hexane/ether 97:3 as eluant
aromatic C), 172.6 [C, C(1)]. HRMS (ES+): Exact mass calculated
for C13H18O2 [M+H]+ 207.1385. Found 207.1393; m/z (ES+) 207.1
{[(C13H18O2)+H]+, 71%}, 202.1 (100%), 161.1 {[(C11H13O)ꢀC2H5O]ꢀ,
48%}, 151.0 (68%), 141.0 (24%).
gave the pure ester ( )-3d (2.94 g, 56%) as a pale yellow oil. vmax
/
cmꢀ1 (film) 2965 (CH), 1737 (CO), 1602, 1454 (Ar), 1152 (C–O);
dH (400 MHz) 0.89 [9H, s, C(CH3)3], 0.99 (3H, t, J 7.1 OCH2CH3)
2.71 [1H, dd, A of ABX, JAB 15.2, JAX 10.9, one of C(2)H2], 2.79 [1H,
dd, B of ABX, JAB 15.2, JBX 5.0, one of C(2)H2], 2.98 [1H, dd, X of
ABX, JAX 10.9, JBX 5.0, C(3)H], 3.85–3.97 (2H, m, OCH2CH3), 7.13–
7.26 (5H, m, ArH).
4.10.8. ( )-Ethyl 3-(4-methoxyphenyl)butanoate ( )-3h53
This was prepared following the procedure described for ( )-3b,
from 3-(4-methoxyphenyl)butanoyl chloride ( )-2h (8.49 g,
39.9 mmol), triethylamine (6.7 mL, 47.88 mmol), dichloromethane
(50 mL) and distilled ethanol (5.8 mL, 99.75 mmol) to yield the
crude ester ( )-3h (6.68 g) as an orange oil. Purification by chroma-
tography on silica gel using hexane/ethyl acetate as eluant (gradi-
ent elution 0–10% ethyl acetate) gave the ester ( )-3h (5.32 g, 56%)
4.10.5. ( )-Ethyl 3-(4-methylphenyl)butanoate ( )-3e24
This was prepared following the procedure described for ( )-3b,
from 3-(4-methylphenyl)butanoyl chloride ( )-2e (12.09 g,
61.47 mmol), triethylamine (10.28 mL, 73.76 mmol), dichloro-
methane (25 mL) and distilled ethanol (8.9 mL, 153.7 mmol) to
yield the crude ester ( )-3e (9.41 g) as a deep orange oil. Purifica-
tion by chromatography on silica gel using hexane/ether 97/3 as
as a clear oil. v
max/cmꢀ1 (film) 2966 (CH), 1732 (CO), 1613, 1513,
1461 (Ar), 1174 (CO); dH (400 MHz) 1.18 (3H, t, J 7.1, OCH2CH3),
1.27 [3H, d, J 7.0, C(4)H3], 2.5 [1H, dd, A of ABX, JAB 14.9, JAX 8.0,
one of C(2)H2], 2.57 [1H, dd, B of ABX, JAB 15.0, JBX 7.2, one of
C(2)H2], 3.17–3.29 [1H, m, X of ABX, C(3)H], 3.77 (3H, s, OCH3),
4.07 (2H, q, J 7.1, OCH2CH3), 6.81–6.85 [2H, m, C(30)H, C(50)H],
7.12–7.16 [2H, m, C(20)H, C(60)H].
eluant gave the pure ester ( )-3e (7.84 g, 62%) as a clear oil. vmax
/
cmꢀ1 (film) 2967 (CH), 1732 (CO), 1516, 1456 (Ar), 1166 (C–O);
dH (400 MHz) 1.19 (3H, t, J 7.1, OCH2CH3), 1.28 [3H, d, J 7.0,
C(4)H3], 2.32 [3H, s, C(40)CH3], 2.51 [1H, dd, A of ABX, JAB 15.0,
JAX 8.2, one of C(2)H2], 2.59 [1H, dd, B of ABX, JAB 15.0, JBX 7.0,
one of C(2)H2], 3.20–3.29 [1H, m, X of ABX, C(3)H], 4.07 (2H, q, J
7.2, OCH2CH3), 7.11 (4H, s, ArH).
4.11. Enzyme screening
4.11.1. General procedure for the hydrolase catalysed kinetic
resolution of the 3-aryl alkanoic ethyl esters ( )-3a–i (analytical
scale)
4.10.6. ( )-Ethyl 3-(3-methylphenyl)butanoate ( )-3f
This was prepared following the procedure described for ( )-3b,
from 3-(3-methylphenyl)butanoyl chloride ( )-2f (7.32 g,
37.2 mmol), triethylamine (6.2 mL, 44.64 mmol), dichloromethane
(30 mL) and distilled ethanol (5.4 mL, 92.99 mmol) to yield the
crude ester ( )-3f (6.53 g) as a dark brown oil. Purification by chro-
matography on silica gel using hexane/ether 97/3 as eluant gave
the pure ester ( )-3f (5.16 g, 67%) as a clear oil. (Found C, 75.13;
A spatula tip of enzyme (ꢁ5-10 mg) was added to the substrate
( )-3a–i (ꢁ50 mg) in 0.1 M phosphate buffer, pH 7 (4.5 mL). Co-
solvents (17% v/v) were added as indicated in Table 3. The reaction
vessel was shaken at 700–750 rpm and incubated at the appropri-
ate temperature for the required length of time. The aqueous layer
was extracted with diethyl ether (3 ꢃ 5 mL) and the combined or-
ganic extracts were filtered through Celite and concentrated under
reduced pressure. The sample was analysed by 1H NMR spectros-
copy, reconcentrated and dissolved in a mixture of hexane/iso-pro-
pyl alcohol (HPLC grade) and enantioselectivity determined by
chiral HPLC. The results of the screen are summarised in the appro-
priate Tables 1–10.
H 8.77. C13H18O2 requires C, 75.69; H, 8.80);
v
max/cmꢀ1 (film)
2968 (CH), 1733 (CO), 1608, 1461 (Ar), 1177 (C–O); dH (400 MHz)
1.18 (3H, t, J 7.1, OCH2CH3), 1.28 [3H, d, J 7.0, C(4)H3], 2.32 [3H,
s, C(30)CH3], 2.51 [1H, dd, A of ABX, JAB 15.0, JAX 8.3, one of
C(2)H2], 2.59 [1H, dd, B of ABX, JAB 15.0, JBX 6.9, one of C(2)H2],
3.19–3.28 [1H, m, X of ABX, C(3)H], 4.07 (2H, q, J 7.1, OCH2CH3),
6.97–7.05 [3H, m, C(40)H, C(50)H, C(60)H, ArH], 7.13–7.19 [1H, m,
C(20)H, ArH]. dC (75.5 MHz) 14.2, 21.5, 21.8 [3 ꢃ CH3, C(4)H3,
OCH2CH3, C(30)CH3], 36.5 [CH, C(3)H], 43.0 [CH2, C(2)H2], 60.3
[CH2, OCH2CH3], 123.7, 127.1, 127.6, 128.4 (4 ꢃ CH, aromatic CH),
138.0, 145.8 (2 ꢃ C, aromatic C), 172.5 [C, C(1)]. HRMS (ES+): Exact
mass calculated for C13H18O2 [M+H]+ 207.1385. Found 207.1390;
m/z (ES+) 207.1 {[(C13H18O2)+H]+, 57%}, 202.1 (100%), 161.1
{[(C11H13O)ꢀC2H5O]ꢀ, 60%}, 151.0 (23%), 141.0 (7%).
4.11.1.1. Preparative scale hydrolysis of ( )-ethyl 3-phenylbut-
anoate ( )-3a. Pseudomonas fluorescens (108 mg) was added to
ethyl 3-phenylbutanoate ( )-3a (510 mg, 2.65 mmol) in 0.1 M
phosphate buffer, pH 7 (20 mL) and this was shaken at 750 rpm
for 64 h at 30 °C. The solution was filtered through a pad of Celite
and the hydrolase washed with water (2 ꢃ 20 mL) and ethyl ace-
tate (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 10% HCl solution and extracted with a further (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 a clear oil (395 mg). Conversion estimated at
50%.34 Purification by column chromatography using hexane/ethyl
acetate as eluant (gradient elution 10–40% ethyl acetate) gave ester
4.10.7. ( )-Ethyl 3-(2-methylphenyl)butanoate ( )-3g
This was prepared following the procedure described for ( )-3b,
from 3-(2-methylphenyl)butanoyl chloride ( )-2g (4.68 g,
23.79 mmol), triethylamine (4 mL, 28.54 mmol), dichloromethane
(20 mL) and distilled ethanol (3.5 mL, 59.46 mmol) to yield the
crude ester ( )-3g (2.85 g) as a yellow oil. Purification by chroma-
tography on silica gel using hexane/ether 97:3 as eluant gave the
ester ( )-3g (2.5 g, 67%) as a clear oil. Further purification by distil-
lation gave the ester ( )-3g (2.14 g, 44%) as a clear oil, bp 96 °C at
0.06 mmHg; (Found C, 75.43; H 8.67. C13H18O2 requires C, 75.69; H,
(R)-3a (178 mg, 35%) as a clear oil ½a D20
¼ ꢀ27:55 (c 1.1, CHCl3), 99%
ꢂ
ee, lit54
½
a 2D5
ꢂ
¼ þ19:0 (c 1.1, CHCl3), (S)-isomer, 90% ee and acid (S)-
1a (147 mg, 34%) as a clear oil ½a D20
¼ þ27:9 (c 1.0, EtOH), 98% ee,
ꢂ
lit55
½
a 2D5
ꢂ
¼ þ24:5 (c 1.0, EtOH), 97% ee. 1H NMR spectra were iden-
8.80%);
v
max/cmꢀ1 (film) 2975 (CH), 1733 (CO), 1605, 1492, 1462
tical to the racemic materials previously prepared.
(Ar), 1175 (CO); dH (400 MHz) 1.17 (3H, t, J7.1, OCH2CH3), 1.25
[3H, d, J 6.9, C(4)H3], 2.37 [3H, s, C(20)CH3], 2.52 [1H, dd, A of
ABX, JAB 15.2, JAX 8.5, one of C(2)H2], 2.61 [1H, dd, B of ABX, JAB
15.2, JBX 6.6, one of C(2)H2], 3.46–3.61 [1H, m, X of ABX, C(3)H],
4.11.1.2. Preparative scale hydrolysis of ( )-ethyl 3-phenylpent-
anoate ( )-3b. Candida antarctica lipase B (immob) (410 mg) was
added to ethyl 3-phenylpentanoate ( )-3b (408 mg, 1.98 mmol) in
0.1 M phosphate buffer, pH 7 (20 mL) and dioxane (17% v/v, 4 mL).
The reaction mixture was shaken at 750 rpm for 62 h at 30 °C, the
solution was filtered through a pad of Celite and the hydrolase
washed with water (2 ꢃ 20 mL) and heptane (10 ꢃ 10 mL). The
4.07 (2H, q,
J 7.1, OCH2CH3), 7.07–7.19 (4H, m, ArH). dC
(75.5 MHz) 14.2, 19.5, 21.3 [3 ꢃ CH3, C(4)H3, OCH2CH3, C(20)CH3],
31.5 [CH, C(3)H], 42.2 [CH2, C(2)H2], 60.3 [CH2, OCH2CH3], 125.1,
126.1, 126.3, 130.4 (4 ꢃ CH, aromatic CH), 135.3, 143.9 (2 ꢃ C,

60
R. E. Deasy et al. / Tetrahedron: Asymmetry 22 (2011) 47–61
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 re-
duced pressure to produce the ester (S)-3b (79.2 mg, 19%) as a light
(gradient elution 10–40% ethyl acetate) gave ester (R)-3f
(105 mg, 22%) as a yellow oil ½a D20
¼ ꢀ24:4 (c 1.0, CHCl3), 94% ee
ꢂ
and acid (S)-1f (107 mg, 26%) as a clear oil ½a D20
¼ þ32:3 (c 0.622,
ꢂ
CHCl3), P99% ee. 1H NMR spectra were identical to the racemic
yellow oil. ½a 2D0
ꢂ
¼ þ9:45 (c 0.55, CHCl3), 65% ee, lit56
½
a 2D6
ꢂ
¼ ꢀ18:3
materials previously prepared.
(c 1.1, CHCl3), (R)-isomer, 97% ee. The aqueous layer was acidified
with 10% HCl solution and extracted with (3 ꢃ 30 mL) ethyl ace-
tate. The combined organic layers were washed with brine
(1 ꢃ 100 mL), dried, filtered and concentrated under reduced pres-
sure to produce the acid (R)-1b (77.6 mg, 22%) as a yellow oil.
4.11.1.7. Preparative scale hydrolysis of ethyl 3-(2-methyl-
phenyl)butanoate ( )-3g. This was prepared following the proce-
dure described for ( )-1a from Pseudomonas fluorescens (74 mg)
and ethyl 3-(2-methylphenyl)butanoate ( )-3g (371 mg,
1.80 mmol) in 0.1 M phosphate buffer, pH 7 (20 mL). The reaction
mixture was shaken at 750 rpm for 66 h at 30 °C to produce a yel-
low oil (268 mg). Conversion estimated at 50%.34 Purification by
column chromatography using hexane/ethyl acetate as eluant (gra-
dient elution 10–40% ethyl acetate) gave ester (R)-3g (100 mg,
½
a 2D0
ꢂ
¼ ꢀ33:7 (c 1.37, C6H6), 90% ee, lit39
½
a 2D5
ꢂ
¼ þ42:3 (c 8.0,
C6H6), (S)-isomer, 83% ee. Conversion estimated at 42%.34 1H
NMR spectra were identical to the racemic materials previously
prepared.
4.11.1.3. Preparative scale hydrolysis of ( )-ethyl 4-methyl-3-
phenylpentanoate ( )-3c. This was prepared following the pro-
cedure described for ( )-3b from Candida antarctica lipase B (im-
mob) (426 mg) and ethyl 4-methyl-3-phenylpentanoate ( )-3c
(428 mg, 1.94 mmol) in 0.1 M phosphate buffer, pH 7 (20 mL).
The reaction mixture was shaken at 750 rpm for 63 h at 30 °C to
27%) as a clear oil ½a D20
¼ ꢀ11:0 (c 1.0, CHCl3), 98% ee and acid
ꢂ
(S)-1g (90 mg, 28%) as a yellow oil ½a D20
¼ þ24:2 (c 1.38, CHCl3),
ꢂ
P99% ee. 1H NMR spectra were identical to the racemic materials
previously prepared.
4.11.1.8. Preparative scale hydrolysis of ( )-ethyl 3-(4-methoxy-
phenyl)butanoate ( )-3h. This was prepared following the pro-
cedure described for ( )-1a from Pseudomonas fluorescens
(100 mg) and ethyl 3-(4-methoxyphenyl)butanoate ( )-3h
(498 mg, 2.24 mmol) in 0.1 M phosphate buffer, pH 7 (20 mL).
The reaction mixture was shaken at 750 rpm for 64 h at 30 °C to
produce an orange oil (428 mg). Conversion estimated at 51%.34
Purification by column chromatography using hexane/ethyl ace-
tate as eluant (gradient elution 10–40% ethyl acetate) gave ester
produce the ester (R)-3c (107 mg, 25%) as a clear oil ½a D20
¼
ꢂ
þ7:05 (c 1.0, CHCl3), 26% ee, lit56
½
a 2D6
ꢂ
¼ ꢀ25:4 (c 1.0, CHCl3), (S)-
isomer, 98% ee and the acid (S)-1c (88 mg, 24%) as a clear oil
½
a 2D0
ꢂ
¼ ꢀ24:35 (c 0.655, CHCl3), 98% ee, lit57
½
a 2D3
ꢂ
¼ þ28:1 (c
1.855, CHCl3), (R)-isomer, 96% ee. Conversion estimated at 21%.34
1H NMR spectra were identical to the racemic materials previously
prepared.
4.11.1.4. Preparative scale hydrolysis of ( )-ethyl 4,4-dimethyl-
3-phenylpentanoate ( )-3d. This was prepared following the
procedure described for ( )-3b from Candida antarctica lipase B
(immob) (200 mg) and ethyl 4,4-dimethyl-3-phenylpentanoate
( )-3d (200 mg, 0.85 mmol) in 0.1 M phosphate buffer, pH 7
(20 mL). The reaction mixture was shaken at 750 rpm for ꢁ65 h
at 35 °C and at 40 °C for the final 24 h to produce the ester (R)-
(R)-3h (212 mg, 43%) as a clear oil ½a D20
¼ ꢀ30:03 (c 1.034, CHCl3),
ꢂ
99% ee and acid (S)-1h (99 mg, 23%) as a yellow oil ½a D20
¼ þ26:25
ꢂ
(c 1.0, EtOH), 97% ee, lit55
½
a 2D5
ꢂ
¼ þ27:5 (c 1.0, EtOH) 94% ee. 1H
NMR spectra were identical to the racemic materials previously
prepared.
4.11.1.9. Preparative scale hydrolysis of ( )-ethyl 3-(4-fluoro-
phenyl)butanoate ( )-3i. This was prepared following the proce-
dure described for ( )-1a from Pseudomonas fluorescens (45 mg)
and ethyl 3-(4-fluorophenyl)butanoate ( )-3i (221 mg, 1.05 mmol)
in 0.1 M phosphate buffer, pH 7 (20 mL). The reaction mixture was
shaken at 750 rpm for 64 h at 30 °C to produce a yellow oil
(171 mg). Conversion estimated at 51%.34 Purification by column
chromatography using hexane/ethyl acetate as eluant (gradient
elution 10–40% ethyl acetate) gave ester (R)-3i (71 mg, 32%) as a
3d (79 mg, 39%) as a clear oil. ½a D20
¼ þ8:0 (c 1.0, CHCl3), 12% ee
ꢂ
and the acid (S)-1d (23 mg, 13%) as a yellow oil which solidified
overnight
½
a 2D0
ꢂ
¼ ꢀ10:5 (c 0.114, CHCl3), 99% ee, lit58
½
a 2D0
ꢂ
¼ ꢀ20:4 (c 2.2, CHCl3), 91% ee. Conversion estimated at
11%.34 1H NMR spectra were identical to the racemic materials pre-
viously prepared.
4.11.1.5. Preparative scale hydrolysis of ( )-ethyl 3-(4-methyl-
phenyl)butanoate ( )-3e. This was prepared following the proce-
dure described for ( )-1a from Pseudomonas cepacia P1 (95 mg) and
ethyl 3-(4-methylphenyl)butanoate ( )-3e (446 mg, 2.26 mmol) in
0.1 M phosphate buffer, pH 7 (20 mL). The reaction mixture was
shaken at 750 rpm for 62 h at 30 °C to produce a yellow oil
(361 mg). Conversion estimated at 49%.34 Purification by column
chromatography using hexane/ethyl acetate as eluant (gradient
elution 10–40% ethyl acetate) gave ester (R)-3e (145 mg, 31%) as
clear oil ½a 2D0
¼ ꢀ24:3 (c 1.0, CHCl3), P99% ee and acid (S)-1i
ꢂ
(67 mg, 35%) as a brown oil ½a D20
¼ þ30:5 (c 1.016, CHCl3), 97%
ꢂ
ee. 1H NMR spectra were identical to the racemic materials previ-
ously prepared.
Acknowledgements
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. Final year project student Christo-
pher O’Donovan is thanked for input on certain aspects of this
work.
a clear oil ½a 2D0
ꢂ
¼ ꢀ28:7 (c 3.5, CHCl3), 97% ee, lit24
½
a 2D5
ꢂ
¼ ꢀ26:2
(c 3.5, CHCl3), 92% ee and acid (S)-1e (163 mg, 40%) as a yellow
oil which solidified overnight ½a D20
¼ þ31:8 (c 1.0, CHCl3), P99%
ꢂ
ee, lit24
½
a 2D5
ꢂ
¼ þ34:2 (c 1.0, CHCl3), 99% ee. 1H NMR spectra were
identical to the racemic materials previously prepared.
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