Preparation of optically pure (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates by Candida parapsilosis ATCC 7330 mediated deracemisation of the racemates

Article abstract of DOI:10.1016/j.tet.2007.02.099

Biocatalytic deracemisation of a range of racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates using Candida parapsilosis ATCC 7330 resulted in pure (S)-enantiomers in yields of up to 80% and ee>99%.

Full text of DOI:10.1016/j.tet.2007.02.099

Tetrahedron 63 (2007) 4126–4133
Preparation of optically pure (3E,5E)-alkyl-2-hydroxy-6-arylhexa-
3,5-dienoates by Candida parapsilosis ATCC 7330 mediated
deracemisation of the racemates
Vaijayanthi Thangavela and Anju Chadhab,
*
aDepartment of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, Tamil Nadu, India
bLaboratory of Bioorganic Chemistry, Department of Biotechnology and National Center for Catalysis Research,
Indian Institute of Technology Madras, Chennai 600 036, Tamil Nadu, India
Received 1 December 2006; revised 8 February 2007; accepted 22 February 2007
Available online 27 February 2007
Abstract—Biocatalytic deracemisation of a range of racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates using Candida parapsilosis
ATCC 7330 resulted in pure (S)-enantiomers in yields of up to 80% and ee>99%.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
which showed activity against Staphylococcus aureus, Es-
cherichia coli, Klebsiella pneumoniae, Bacillus subtilis and
Mycobacterium smegmatis,13 and Lachnellins B (2, Fig. 1)
and C (3, Fig. 1) isolated from submerged cultures of the
Lachnellula ascomycete A,14 which are inhibitors of malate
synthase and show high cytotoxic and antimicrobial activi-
ties. These multifunctional chiral synthons are important
building blocks in the synthesis of many bioactive mole-
cules, e.g., 2(3H)-benzofuranone-3,5-dihydroxy15 and an
antitumour agent topotecin16 in addition to other clinical
applications.17 Ruthenium catalyzed asymmetric reduction
of (3E,5E)-isopropyl-2-oxo-6-phenylhexa-3,5-dienoate re-
quires stringent conditions to give the hydroxy compound
with an ee of only 7%.18 Given the multifunctional groups
on these molecules, it is not easy to design chemical methods
for their preparation. Biocatalysts on the other hand, which
are known for their specificity, can be used very effectively
for such reactions as shown by the work reported herein.
A series of (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-di-
enoates were prepared in high ee (up to >99%) and yields
(up to 80%) using C. parapsilosis ATCC 7330, which
deracemised these racemates to single enantiomers. Signifi-
cantly, whole cells were used for this purpose thus obviating
the need for added cofactors and elaborate enzyme purifica-
tion protocols. We report here the synthesis of the racemic
substrates, i.e., (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-
dienoates, their chiral separation using chiral HPLC,
optimization of the C. parapsilosis ATCC 7330 mediated
deracemisation reaction for the representative substrate race-
mic(3E,5E)-ethyl-2-hydroxy-6-phenylhexa-3,5-dienoateand
substrate tolerance of the biocatalyst with structurally differ-
ent (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates.
As a strategy to prepare optically pure organic molecules,
deracemisation1,2 has several advantages over resolution,3,4
especially when a single biocatalyst5,6 can be employed for
the purpose. In continuation of our work on Candida para-
psilosis ATCC 7330 mediated deracemisation of a- and b-
hydroxy esters,7–12 we now report here for the first time, the
biocatalytic preparation of optically pure (3E,5E)-alkyl-2-
hydroxy-6-arylhexa-3,5-dienoates. Though the syntheses
of these optically pure (3E,5E)-alkyl-2-hydroxy-6-arylhexa-
3,5-dienoates except for (3E,5E)-isopropyl-2-hydroxy-6-
phenylhexa-3,5-dienoate are not reported yet, compounds
containing this important subunit, i.e., b, g, d, u unsaturated
a-hydroxy carbonyl, have been found to have cytotoxic and
antibacterial activities.13,14 Some well known examples are
avellaneol (1, Fig. 1), isolated from Hypocrea avellanea,
O
HO
H
H
HO
H
Me
Me MeO
OH
Me
MeO
O
O
Me
O
1 (Avellaneol)
2 (Lachnellins B)
3 (Lachnellins C)
Figure 1. Examples of b, g, d, u unsaturated a-hydroxy carbonyl com-
pounds.
Keywords: (3E,5E)-Alkyl-2-hydroxy-6-arylhexa-3,5-dienoates; Synthesis;
Biocatalytic deracemisation; Candida parapsilosis.
* Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102;
e-mail: anjuc@iitm.ac.in
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.02.099

V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
4127
2. Results and discussion
2.1. Synthesis of substrates
3,5-dienoates (8a–8k, Scheme 1) in moderate to good yields
(68–85%). Reduction of (3E,5E)-alkyl-2-oxo-6-arylhexa-
3,5-dienoates (7a–7k, Scheme 1) using sodium borohydride
is a simple method and it does not give the transesterified
product, which was observed in the case of b-oxo esters.25
Racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates
(Fig. 2) were synthesized by reported procedures to get
high yields of the target esters. A representative scheme is
shown in Scheme 1 and a detailed discussion follows. Aryl
substituted cinnamaldehydes (5a–5k, Scheme 1) were pre-
pared in good yields (60–70%) as per the reported method19
and condensed with pyurvate.20 (3E,5E)-2-Oxo-6-arylhexa-
3,5-dienoic acids (6a–6k, Scheme 1) were obtained in 55–
75% yields. As per the reported method,21 (3E,5E)-ethyl
2-oxo-6-phenylhexa-3,5-dienoate (7a, Scheme 1) was syn-
thesized via a two-step procedure (86% yield) consisting of
the boron trifluoride-promoted reaction of 2-(trimethyl-
siloxy) acrylic esters with acetals followed by treatment
with silica gel in benzene under reflux. In order to avoid
these harsh reaction conditions and hazardous reagents, var-
ious (3E,5E)-alkyl-2-oxo-6-arylhexa-3,5-dienoates (7a–7k,
Scheme 1) were prepared by esterification of the correspond-
ing acids using a heteropoly acid9,22,23 as a catalyst. Sodium
borohydride reduction of 2-oxo esters24 resulted in the cor-
responding racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-
2.2. Chiral separation of racemic (3E,5E)-alkyl-2-hy-
droxy-6-arylhexa-3,5-dienoates (8a–8k) by chiral HPLC
The direct separation of enantiomers of (3E,5E)-alkyl-
2-hydroxy-6-arylhexa-3,5-dienoates (8a–8k, Table 1) was
achieved on a chiral stationary phase (CSP) column on
HPLC. Compounds 8a–8c, 8e, 8g, and 8j–8k were resolved
using a cellulose tris(3,5-dimethylphenyl)carbamate (Chir-
alcel OD-H) column and 8d, 8f–8i were resolved using a
cellulose tris(4-methyl)benzoate (Chiralcel OJ-H) column.
These CSPs have also been used to separate other a- and
b-hydroxy esters.9,12 Enantiomeric separation of 8a–8k
Table 1. Chiral separation of racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-
3,5-dienoates (8a–8k) by chiral HPLC
S. no
C. noa
Column used
Hex:IPA
Retention time (min)
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
8g
8h
8i
OD-H
OD-H
OD-H
OJ-H
OD-H
OJ-H
OD-H
OJ-H
OJ-H
OD-H
OD-H
98:2
99:1
98:2
98:2
98:2
98:2
97:3
97:3
98:2
98:2
98:2
20.3; 25.2
35.5; 40.5
20.6; 25.8
25.9; 28.5
23.0; 24.8
26.4; 30.6
36.7; 41.3
46.5; 66.0
22.2; 26.8
12.4; 15.2
20.2; 27.8
OH
O
R'
O
"RS"
R
9
10
11
R = H, o-Me, p-Me, o-Cl, p-Cl, o-NO2
R’= CH3, CH3CH2, CH3CHCH3, CH2C6H5
8j
8k
a
C. no.—compound number.
Figure 2.
O
O
O
O
O
O-Na+
OH
H
H
O
O
KOH,
Aq CH3OH(50%
0-10°C, 3-4 hrs
O
Ba(OH)2
,
THF
R
R
R
Yield 55-75%
6a - 6k
Yield 60-70%
5a - 5k
70°C,
3-5 hrs
)
4a- 4k
R'OH,
PWA , 80-90°C
6-8 hrs
O
OH
O
O
NaBH4
EtOH
0-10 °C,
R'
R'
O
O
R
R
Yield 60-74%
Yield 70-85%
8a- 8k
15-20 minutes
7a- 7k
R; R’
a : H, CH3CH2
b : H, CH3
c: o-Me, CH3CH2
,
d: p-Me, CH3CH2
e: p-Me, CH3
f: p-Cl, CH3CH2
g : o-NO2
,
CH3CH2
CH3
h: o-NO2
,
i: o-Cl, CH3CH2
j :H, CH3CHCH3
k : H, CH2C6H5
Scheme 1. Synthesis of (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates (8a–8k).

4128
V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
was achieved by varying the composition of hexane/isopro-
panol in the range of 99:1–97:3 and is reported here for the
first time (Table 1).
100
80
60
40
20
0
80
60
40
20
0
2.3. Deracemisation of racemic (3E,5E)-alkyl-2-hy-
droxy-6-arylhexa-3,5-dienoates (8a–8k) mediated
by whole cells of C. parapsilosis ATCC 7330
Deracemisation is one of the most efficient methods to
produce chiral synthons since it delivers 100% of a single
enantiomer from its racemate.1 Deracemisation of racemic
N-(1-hydroxy-1-phenylethyl)benzamide to the (R)-enantio-
mer (98% ee) using whole cells of Cunninghamella echinu-
lata NRRL 1384 is known.26 Highly enantioselective
conversion of the racemic 1-phenyl-1,2-ethanediol by stereo-
inversion involving a cofactor-dependent oxidoreductase
system of C. parapsilosis CCTCC M203011 gave (S)-1-
phenyl-1,2-ethanediol in 96.7% ee and 96% yield.27 A com-
bination of lipase and ruthenium metal was used for the
dynamic kinetic resolution of racemic, secondary allylic al-
cohols, and a-hydroxy esters in high optical purity (94% ee)
and high yield (76%),28–30 in which ruthenium metal race-
mizes the unwanted enantiomers in situ.31 Whole cells of
C. parapsilosis ATCC 7330 have proven to be very effective
biocatalysts for the deracemisation of a- and b-hydroxy
esters as reported by us earlier.9,12 We have also used this
biocatalyst for asymmetric reduction of a-keto esters.8 In
the present study, C. parapsilosis ATCC 7330 has been
employed to deracemise a-hydroxy esters with a conjugated
diene system. Formation of the ‘S’ enantiomers with high ee
(up to >99%) and yields (up to 80%) reveal the wide sub-
strate acceptance of this biocatalyst.
Enantiomeric excess (%)
Yield (%)
0
2
4
Time (h)
Figure 3. Time course of the deracemisation of (3E,5E)-ethyl-2-hydroxy-6-
phenylhexa-3,5-dienoate (8a).
Deracemisation of (RS)-8a (3 mg mlꢁ1) by C. parapsilosis
ATCC 7330 was carried out at 15, 20, 25, 30, 37, 40, and
45 ꢀC under otherwise identical conditions (i.e., 1.0 g wet
cells per 1 ml; 0.75 ml of ethanol for 3 h) (Fig. 4). The ee
and isolated yield increased with increasing temperature
from 15 ꢀC (88% ee, 68% yield) to 25 ꢀC (>99% ee, 80%
yield) but decreased at 30 ꢀC (96% ee, 74% yield) and
45 ꢀC (8% ee, 58% yield). This variation in the ee with
respect to temperature could be due to the different thermal
stability of the ketoreductases encoded within the genome.34
The deracemisation carried out at 25 ꢀC gave the best results
(yieldw81% and >99% ee).
2.4.3. Screening of solvents. The wet cell mass of C. para-
psilosis ATCC 7330 used for deracemisation reactions is
suspended in aqueous medium but the substrate being an or-
ganic molecule, has limited solubility in water. Therefore
the substrate had to be dissolved in an organic solvent,
2.4. Optimization study for the deracemisation of
(3E,5E)-ethyl-2-hydroxy-6-phenylhexa-3,5-dienoate
(8a) using C. parapsilosis ATCC 7330
The representative dienoate used for optimizing the para-
meters for deracemisation was (3E,5E)-ethyl-2-hydroxy-6-
phenylhexa-3,5-dienoate (8a, Scheme 2) and the product
was monitored for its ee (%) and yield (%).
82
80
78
100
76
74
72
70
68
66
64
62
60
58
56
54
80
60
40
20
0
2.4.1. Time course. The time of the deracemisation was
typically 3 h, which is within the range (1.5–4 h) reported
for the deracemisation of racemic alkyl 2-hydroxy-4-aryl-
but-3-enoates.9 As can be seen from Figure 3, the reaction
mixture was analyzed at regular time intervals of 30 min
for 4.5 h. After 3 h (S)-9a was obtained in >99% ee and
80% isolated yield.
Enantiomeric excess(%)
Yield(%)
2.4.2. Effect of temperature. Most of the biotransforma-
tions carried out by resting cells of C. parapsilosis CCTCC
M203011 (China Center for Type Culture Collection) were
done at 30 ꢀC32,33 but deracemisation of racemic alkyl
2-hydroxy-4-arylbut-3-enoates by C. parapsilosis ATCC
7330 were reported at 25 ꢀC from our own laboratory.9
15
20
25
30
35
40
45
Temperature (°C)
Figure 4. Effect of temperature in the deracemisation of (3E,5E)-ethyl-2-
hydroxy-6-phenylhexa-3,5-dienoate (8a).
OH
HO
H
Candida
parapsilosis ATCC 7330
O
O
'S'
O
O
'RS'
99% ee
Scheme 2. Optimization study for (3E,5E)-ethyl-2-hydroxy-6-arylhexa-3,5-dienoate (8a).

V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
4129
preferably miscible with water. The effect of pure solvents
(partition coefficient, log P) on the deracemisation of 8a cat-
alyzed by wet cells of C. parapsilosis ATCC 7330 was stud-
ied by monitoring the ee (%) and yield (%) of the product
(Fig. 5). The reaction mixture of 3 mg of racemic substrate
(8a) dissolved in 75 ml of pure solvent was added to 1.0 g
of wet cells of C. parapsilosis ATCC 7330 suspended in
0.9 ml of distilled water and incubated for 3 h at 25 ꢀC and
150 rpm. After completion of the reaction, product was
extracted using ethyl acetate (1 ml) and ee was determined
using chiral HPLC. The use of protic solvents like methanol
(log P ꢁ0.74) and ethanol (log P ꢁ0.3) for dissolving the
substrate for deracemisation resulted in good chemical
(w77–79%) and optical yields (>99% ee). However, in the
case of solvents like benzene (log P 2.13), toluene (log P
2.5), and dichloromethane (log P 1.25), the ee of the product
was poor (2–10%) as were the chemical yields (47–57%).
In the case of solvents like diethylether (log P ꢁ0.01159),
tetrahydrofuran (log P 0.49), and 1,4-dioxane (log P ꢁ0.42),
deracemisation of 8a resulted in moderate chemical (w25–
69%) and optical yields (34–94% ee). Optical and chemical
yields of the deracemised product of 8a therefore seem to in-
crease as log P decreases. This is also true in the case of de-
racemisation of 2-hydroxy-4-aryl-butanoic esters mediated
by C. parapsilosis ATCC 7330.24 Alcohols are efficient sol-
vents as they are also substrates for enzymes, especially
alcohol dehydrogenases, which can regenerate cofactors.
This is advantageous for the deracemisation process, which
involves oxidation and reduction35 and was also observed in
the asymmetric reduction of ketones using secondary alco-
hols (2-propanol) using Rhodococcus ruber DSM 44541.36
The solvent used for the biotransformations in this investiga-
tion was ethanol.
2.5. Substrate specificity
Under the optimized reaction conditions, racemic (3E,5E)-
alkyl-6-aryl-2-hydroxyhexa-3,5-dienoates (8a–8k) were de-
racemized into their (S)-enantiomers on incubation at 25 ꢀC
for 3 h with whole cells of C. parapsilosis ATCC 7330
(Scheme 3) and the results obtained are shown in Table 2.
100
80
60
40
20
0
Enantiomeric excess (%)
Yield (%)
The most plausible pathway for the deracemisation of a-
hydroxy esters involves stereoinversion mediated by oxido-
reductases.9a Deracemisation of methyl and ethyl esters of
aryl unsubstituted (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-
3,5-dienoates (8a–8b, Table 2) by whole cells of C. parapsi-
losis ATCC 7330 resulted in the optically pure (S)-enantio-
mers in excellent isolated chemical (73–80%) and optical
yields (93–99% ee). These results show that the biocatalyst
C. parapsilosis ATCC 7330 accepts these dienoates as well
as the (E)-2-hydroxy-4-arylbut-3-enoic esters (98–99% ee
and 74–75% yields) reported earlier.9 Among the other
aryl unsubstituted compounds with branched and benzyl
esters, deracemisation of (3E,5E)-isopropyl-6-phenyl-2-hy-
droxyhexa-3,5-dienoate (8j, Table 2) results in the formation
of the (S)-enantiomer in excellent ee (98% ee) and moderate
chemical yield (65%) while aryl unsubstituted (3E,5E)-
benzyl-6-phenyl-2-hydroxyhexa-3,5-dienoate (8k, Table 2)
gives the deracemised product in only 42% ee and 51% yield.
Introduction of electron donating methyl groups at ortho or
para positions in (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-3,5-
dienoates (8c–8e, Table 2) causes a decrease in both isolated
Figure 5. Screening of pure solvents for the deracemisation of (3E,5E)-
ethyl-2-hydroxy-6-phenylhexa-3,5-dienoate (8a). [Experimental detail:
3 mg of racemic substrate (8a) dissolved in 75 ml of pure solvent was added
to 1.0 g of wet cells of C. parapsilosis ATCC 7330 suspended in 0.9 ml of
distilled water. Incubated for 3 h at 25 ꢀC and 150 rpm. Product was
extracted using ethyl acetate (1 ml) and ee was determined using Chiralcel
OD-H column, solvent: hexane/isopropanol 98:2, flow rate: 1 ml minꢁ1].
OH
O
HO
H
O
Candida
R'
R'
parapsilosis ATCC 7330
O
O
'S'
'RS'
3 h, 25 °C
R
R
80-99% ee
8a-8k
9a-9k
R, R’
a: H, CH3CH2
b: H, CH3
c: o-Me, CH3CH2
,
d: p-Me, CH3CH2
e: p-Me, CH3
f: p-Cl, CH3CH2
g: o-NO2
h: o-NO2
,
CH3CH2
CH3
,
i: o-Cl, CH3CH2
j: H, CH3CHCH3
k: H, CH2C6H5
Scheme 3. Deracemisation of racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates (8a–8k).

4130
V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
Table 2. Deracemisation of various racemic (3E,5E)-alkyl-2-hydroxy-6-
arylhexa-3,5-dienoates (8a–8k) using Candida parapsilosis ATCC 7330
control experiment reveals that 10 is formed as a result of
biocatalytic hydrolysis. Compound 10 was isolated and
its specific rotation ([a]2D5 +92.0 (c 1.0, CH3OH) was com-
pared with the literature value37 giving an ee of w99%
indicating that in all likelihood enzymatic hydrolysis follows
deracemisation.
Entry
R
R0
ee
(%) (%)
Yield [a]2D5
Abs.
Conf
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
H
Et
Me
>99 80
93 73
82 65
95 74
87 72
>99 55
90 66
91 69
85 70
98 65
42 51
+92.0 (c 1.0, CH3OH)a,b
+92.1 (c 1.0, CH3OH)a,b
+3.4 (c 1.0, CHCl3)
+2.7 (c 1.3, CHCl3)
+5.1 (c 1.1, CHCl3)
59.6 (c 1.2, CH3OH)a,b
Nd
S
S
o-Me Et
p-Me Et
p-Me Me
p-Cl
o-NO2 Et
o-NO2 Me
o-Cl
H
H
Nd
Nd
Nd
S
Nd
Nd
Nd
S
3. Conclusion
Et
A convenient and simple procedure for the synthesis of
racemic (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates
from easily available starting materials with improvisations
is reported. The synthesized racemic esters were resolved
using chiral HPLC on chiral columns OD-H and OJ-H
using hexane and isopropyl alcohol as eluents. Whole
cells of C. parapsilosis ATCC 7330 were used to catalyze
the deracemisation of aryl unsubstituted and substituted
(3E,5E)-alkyl-2-hydroxy-6-aryl-hexa-3,5-dienoates to give
the corresponding optically pure (S)-dienoates (42 to
>99% ee and 51–80% yields). It has been found that C. par-
apsilosis ATCC 7330 mediated deracemisation of (i) aryl
substituted (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-3,5-dienoates
that have either electron withdrawing or electron donating
substituents at ortho/para positions in the aromatic ring give
lower chemical yields (51–74%) and optical purity (42–
99%) as compared to aryl unsubstituted dienoates (chemical
yields 73–80% and optical purity 93–99%) and (ii) aryl
unsubstituted and aryl substituted (electron releasing/
withdrawing) (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-3,5-di-
enoates give moderate to good isolated yields (51–80%)
but moderate to excellent optical purity (42–99%) as com-
pared to (E)-alkyl-2-hydroxy-4-arylbut-3-enoic esters (iso-
lated yield 66–93% and ee 91–99%).9
Nd
Nd
Et
i-pr
Benzyl
8j
8k
+90.0 (c 1.0, CH3OH)a,b
+93.2 (c 1.0, CH3OH)a,b
S
Nd: Not determined.
Ref. 37.
Specific rotations were measured after hydrolysis.
a
b
chemical (65–74%) and optical yields (82–95% ee). This
was also seen in aryl substituted (E)-2-hydroxy-4-arylbut-
3-enoic esters, where the introduction of electron donating
groups caused a decrease in optical yields (90–92% ee)
and isolated chemical yields (68–70%). In aryl ortho-
substituted
(3E,5E)-alkyl-6-aryl-2-hydroxyhexa-3,5-di-
enoate (8c, Table 2), as compared to its para substituted
counterpart (8d, Table 2), the chemical yield is marginally
less (65%) probably due to prominent steric effects in the
ortho compound. Introduction of a chloro group at the ortho
position (8i, Table 2), which has both a mesomeric effect
(+ve) and an inductive effect (ꢁve), gives marginally higher
chemical (70%) and lower optical yields (85% ee) as com-
pared to the para substituted dienoate (8f, Table 2) [chemical
(55%) and optical yield (>99% ee)] as seen earlier in (E)-2-
hydroxy-4-arylbut-3-enoic esters, which give chemical (65–
68%) and optical yield (95–98% ee).9 The presence of strong
electron withdrawing nitro groups at the ortho position in the
aromatic ring of dienoates (8g–8h, Table 2) produced the
optically pure product in 66–69% yield and 90–91% ee
as reported for meta substituted (E)-2-hydroxy-4-arylbut-
3-enoic esters (93–95% ee and 69–70% yield).9 Aryl
substituted racemic (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-
3,5-dienoates (8c–8i, Table 2) give lower chemical yields
(51–74%) and optical purity (42–99%) as compared to aryl
unsubstituted dienoates (8a–8b, Table 2) (chemical yields
73–80% and optical purity 93–99%).
4. Experimental
4.1. General methods
C. parapsilosis ATCC 7330 was purchased from American
Type Culture Collection, Manassas, VA 20108, USA. All
chemicals used for media preparation were purchased
locally. All substrates were synthesized using the given
schemes. 1H and 13C NMR spectra were recorded in CDCl3
solution on a JEOL GSX400 and Bruker AV-400 spectrom-
eters operating at 400 MHz. Chemical shifts are expressed in
parts per million values using TMS as an internal standard.
Infrared spectra were recorded on a Shimadzu IR 470 Instru-
ment. Mass spectra were recorded on a Q TOF micromass
The deracemisation of racemic (3E,5E)-ethyl-6-phenyl-2-
hydroxyhexa-3,5-dienoate (8a) competes with the formation
of small amount (20%) of the corresponding (3E,5E)-6-
phenyl-2-hydroxyhexa-3,5-dienoic acid (10, Scheme 4). A
OH
H
HO
'S'
Candida parapsilosis
O
O
ATCC 7330
25 °C, 3 hrs
O
O
9a
8a
+
HO
H
OH
OH
OH
or
O
O
'S'
'RS'
10
Scheme 4. Partial hydrolysis of the substrate (3E,5E)-ethyl-2-hydroxy-6-arylhexa-3,5-dienoate (8a) during deracemisation.

V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
4131
spectrometer. The ee (ee%) was determined by HPLC anal-
ysis. HPLC analysis was done on a Jasco PU-1580 liquid
chromatograph equipped with PDA detector. The chiral
columns used were Chiralcel OD-H and Chiralcel OJ-H
(Daicel, 4.6ꢂ250 mm). The solvent used was hexane/iso-
propanol (99:1, 98:2, and 97:3) at a flow rate of 1 ml minꢁ1
and the absorbance was monitored using a PDA detector at
254 nm. Optical rotations were determined on an AutopalÒ
digital polarimeter. Melting points were determined on
Toshniwal melting point apparatus and are uncorrected.
TLC was done using Kieselgel 60 F254 aluminum sheets
(Merck 1.05554).
5.3 Hz, 1H), 6.57 (dd, J¼15.6, 10.4 Hz, 1H), 6.58 (d,
J¼15.6 Hz, 1H), 6.75 (dd, J¼15.6, 10.4 Hz, 1H), 7.23–
7.40 (5H, m); 13C NMR (400 MHz, CDCl3) d: 14.1, 62.2,
71.0, 126.4, 127.5, 127.7, 128.6, 129.0, 132.5, 133.8, 136.9,
173.2; IR nmax (KBr): 3367, 2986, 2939, 1741, 1732, 1638,
1448, 1289, 1092, 1054, 989 cmꢁ1; HRMS (ESI): found
255.1007, C14H16O3Na [M+Na]+ requires 255.0997.
4.4.2. (3E,5E)-Methyl-2-hydroxy-6-phenylhexa-3,5-dien-
1
oate (8b). Yellow solid; mp 33 ꢀC; H NMR (400 MHz,
CDCl3) d: 3.25 (br s, 1H), 3.80 (s, 3H), 4.77 (d, J¼5.6 Hz,
1H), 6.19 (dd, J¼15.2, 5.6 Hz, 1H), 6.59 (dd, J¼15.2,
10.7 Hz, 1H), 6.61 (d, J¼15.2 Hz, 1H), 6.77 (dd, J¼15.2,
10.7 Hz, 1H), 7.22–7.39 (m, 5H); 13C NMR (400 MHz,
CDCl3) d: 50.9, 60.4, 71.1, 126.5, 127.4, 127.8, 128.6,
128.9, 134.0, 136.8, 173.4; IR nmax (KBr): 3558, 2920,
4.2. Synthesis of (3E,5E)-alkyl-2-hydroxy-6-arylhexa-
3,5-dienoates (8a–8k)
Reduction of (3E,5E)-ethyl-2-oxo-6-phenylhexa-3,5-dien-
oate (7a) (0.17 mmol, 400 mg) was carried out with sodium
borohydride (0.17 mol, 6 mg) using ethanol as solvent (5 ml)
for 20 min at 0–10 ꢀC. After the reaction was complete the
reaction mixture was neutralized with dil HCl, which was
extracted with ethyl acetate (3ꢂ10 ml). The collected
organic fractions were dried and concentrated. Compound
(3E,5E)-ethyl-2-hydroxy-6-phenylhexa-3,5-dienoate (8a)
was obtained as a yellow solid in 81% yield (326 mg,
0.14 mmol) after column purification using hexane/ethyl
acetate (95:5) as solvent. The same procedure was followed
for compounds 8b–8k (Table 1).
2350, 2335, 1772, 1742, 1507, 1460, 974, 761, 700 cmꢁ1
;
HRMS (ESI): found 219.1012, C13H15O3 [M+H]+ requires
219.1021.
4.4.3. (3E,5E)-Ethyl-2-hydroxy-6-(o-methylphenyl)hexa-
3,5-dienoate (8c). Yellow solid; mp 68 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 1.48 (t, J¼6.8 Hz, 3H), 2.3 (s, 3H),
3.14 (br s, 1H), 4.27 (q, J¼6.8 Hz, 2H), 4.79 (d, J¼5.3 Hz,
1H), 6.18 (dd, J¼15.6, 5.3 Hz, 1H), 7.04–7.19 (m, 4H),
7.12 (d, J¼15.6 Hz, 1H), 7.27 (dd, J¼15.6, 10.4 Hz, 1H),
7.51 (dd, J¼15.6, 10.4 Hz, 1H); 13C NMR (400 MHz,
CDCl3) d: 14.1, 21.1, 71.3, 126.2, 126.8, 127.0, 127.5,
128.4, 128.9, 129.5, 129.7, 130.4, 133.4, 134.4, 173.0; IR
nmax (KBr): 3465, 3060, 2981, 2932, 1732, 1693, 1594,
1486, 1460, 1374, 1264, 1136, 1083, 1023, 974, 861, 738,
701 cmꢁ1; HRMS (ESI): found 247.1333, C15H19O3
[M+H]+ requires 247.1334.
4.3. Deracemisation of racemic (3E,5E)-alkyl-2-
hydroxy-6-arylhexa-3,5-dienoates (8a–8k)
4.3.1. Culture medium for the growth of C. parapsilosis
ATCC 7330. Cells of the yeast, C. parapsilosis ATCC
7330 were grown as reported earlier9 and harvested after
44 h and used for the deracemisation reaction.
4.4.4. (3E,5E)-Ethyl-2-hydroxy-6-(p-methylphenyl)hexa-
3,5-dienoate (8d). Yellow liquid; 1H NMR (400 MHz,
CDCl3) d: 1.28 (t, J¼7.0 Hz, 3H), 2.33 (s, 3H), 3.9 (br s,
1H), 4.26 (q, J¼7.0 Hz, 2H), 4.76 (d, J¼5.3 Hz, 1H), 5.83
(dd, J¼15.2, 5.3 Hz, 1H), 6.57 (d, J¼15.2 Hz, 1H), 6.66
(dd, J¼15.2, 10.7 Hz, 1H), 6.74 (dd, J¼15.2, 10.7 Hz,
1H), 7.12 (d, J¼7.9 Hz, 2H), 7.28 (d, J¼7.9 Hz, 2H); 13C
NMR (400 MHz, CDCl3) d: 14.1, 21.1, 61.5, 71.1, 124.5,
126.5, 126.6, 128.5, 129.3, 132.7, 133.4, 137.7, 173.4; IR
nmax (KBr): 3477, 2981, 2923, 1731, 1597, 1513, 1446,
1371, 1259, 1207, 1079, 1018, 858, 716, 488, 451, 438,
411 cmꢁ1; HRMS (ESI): found 247.1335, C15H19O3
[M+H]+ requires 247.1334.
4.3.2. A typical procedure for the deracemisation reac-
tion of 8a using whole cells of C. parapsilosis ATCC
7330. Deracemisation of racemic (3E,5E)-ethyl-2-hydroxy-
6-phenylhexa-3,5-dienoate (8a) was done as reported earlier
except for the quantity (1.0 g mlꢁ1 wet weight) of biocata-
lyst C. parapsilosis ATCC 7330 and the time course of the
reaction (3 h).9 After incubation, the products were extracted
into ethyl acetate, concentrated, and analyzed for enantio-
meric purity. Appropriate control experiments were carried
out using (i) cells in the reaction mixture without the sub-
strate and (ii) substrate in the reaction mixture without cells.
The HPLC profile of the control experiments revealed that
(i) nothing from the cells on extraction co-eluted with the
product and (ii) the substrate did not yield any products in
water. In order to determine the isolated chemical yield,
the deracemisation of 8a was carried out with 72 mg of the
substrate. The other racemic (3E,5E)-alkyl-2-hydroxy-6-
arylhexa-3,5-dienoates (8b–8k) were also used as substrates
in the same manner.
4.4.5. (3E,5E)-Methyl-2-hydroxy-6-(p-methylphenyl)-
1
hexa-3,5-dienoate (8e). Yellow solid; mp 77 ꢀC; H NMR
(400 MHz, CDCl3) d: 2.28 (s, 3H), 2.95 (br s, 1H), 3.74 (s,
3H), 4.69 (d, J¼6.0 Hz, 1H), 5.73 (dd, J¼15.2, 6.0 Hz,
1H), 6.51 (dd, J¼15.2, 10.8 Hz, 1H), 6.50 (d, J¼15.2 Hz,
1H), 6.65 (dd, J¼15.2, 10.8 Hz, 1H), 7.05 (d, J¼8.0 Hz,
2H), 7.22 (d, J¼8.0 Hz, 2H); 13C NMR (400 MHz, CDCl3)
d: 21.2, 52.9, 71.1, 126.4, 127.6, 128.2, 128.5, 129.3,
129.5, 132.9, 137.8, 173.7; IR nmax (KBr): 3432, 2359,
1732, 1677, 1578, 1513, 1439, 1279, 1080, 1020,
811 cmꢁ1; HRMS (ESI): found 255.0999, C14H16O3Na
[M+Na]+ requires 255.0997.
4.4. Analytical data for the substrates (8a–8k)
4.4.1. (3E,5E)-Ethyl-2-hydroxy-6-phenylhexa-3,5-dien-
1
oate (8a). Yellow solid; mp 37 ꢀC; H NMR (400 MHz,
CDCl3) d: 1.3 (t, J¼6.9 Hz, 3H), 3.06 (br s, 1H), 4.2 (q,
4.4.6. (3E,5E)-Ethyl-2-hydroxy-6-(p-chlorophenyl)hexa-
3,5-dienoate (8f). Yellow solid; mp 90 ꢀC; 1H NMR
J¼6.9 Hz, 2H), 4.74 (d, J¼5.3 Hz, 1H), 5.83 (dd, J¼15.6,

4132
V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
(400 MHz, CDCl3) d: 1.28 (t, J¼6.8 Hz, 3H), 3.09 (br s, 1H),
4.26 (q, J¼6.8 Hz, 2H), 4.76 (d, J¼5.3 Hz, 1H), 5.83 (dd,
J¼15.2, 5.3 Hz, 1H), 6.57 (d, J¼15.2 Hz, 1H), 6.66 (dd,
J¼15.2, 10.6 Hz, 1H), 6.74 (dd, J¼15.2, 10.6 Hz, 1H),
7.12 (d, J¼7.9 Hz, 2H), 7.28 (d, J¼7.9 Hz, 2H); 13C NMR
(400 MHz, CDCl3) d: 14.1, 62.1, 71.0, 124.5, 126.4, 126.6,
128.5, 129.3, 132.7, 133.8, 134.2, 173.4; IR nmax (KBr):
3482, 2989, 2936, 2365, 2335, 1742, 1597, 1483, 1392,
1263, 1088, 822 cmꢁ1; HRMS (ESI): found 289.0618,
C14H15O3ClNa [M+Na]+ requires 289.0607.
4.4.11. (3E,5E)-Benzyl-2-hydroxy-6-phenylhexa-3,5-di-
enoate (8k). Yellow solid; mp 118 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.09 (br s, 1H), 4.79 (d, J¼5.4 Hz,
1H), 5.23 (q, J¼9.7 Hz, 2H), 5.85 (dd, J¼15.6, 5.4 Hz,
1H), 6.59 (dd, J¼15.6, 10.7 Hz, 1H), 6.75 (dd, J¼15.6,
10.7 Hz, 1H), 6.57 (d, J¼15.6 Hz, 1H), 7.21–7.39 (m,
10H); 13C NMR (400 MHz, CDCl3) d: 67.7, 71.7, 126.5,
126.9, 127.4, 127.8, 128.3, 128.5, 128.6, 128.7, 132.7,
134.0, 134.9, 136.9, 173.1; IR nmax (KBr): 3418, 3063,
2936, 1957, 1887, 1731, 1581, 1496, 1385, 1270, 1210,
970, 847, 750, 496, 452, 413 cmꢁ1; HRMS (ESI): found
317.1140, C19H18O3Na [M+Na]+ requires 317.1154.
4.4.7. (3E,5E)-Ethyl-2-hydroxy-6-(o-nitrophenyl)hexa-
3,5-dienoate (8g). Yellow liquid; 1H NMR (400 MHz,
CDCl3) d: 1.24 (t, J¼7.3 Hz, 3H), 3.04 (br s, 1H), 4.20 (q,
J¼7.3 Hz, 2H), 4.69 (d, J¼5.3 Hz, 1H), 5.89 (dd, J¼15.1,
5.3 Hz, 1H), 6.57 (dd, J¼15.1, 10.7 Hz, 1H), 6.66 (dd,
J¼15.1, 10.7 Hz, 1H), 6.98 (d, J¼15.1 Hz, 1H), 7.28 (t,
J¼7.8 Hz, 1H), 7.46 (t, J¼7.8 Hz, 1H), 7.55 (d, J¼7.8 Hz,
1H), 7.81 (d, J¼7.8 Hz, 1H); 13C NMR (400 MHz, CDCl3)
d: 14.3, 65.5, 71.1, 124.9, 128.1, 128.2, 128.3, 131.9,
132.1, 132.6, 132.8, 133.1, 148.0, 173.1; IR nmax (neat):
3501, 1734, 1644, 1521, 990, 784, 740 cmꢁ1; HRMS
(ESI): found 300.0841, C14H15NO5Na [M+Na]+ requires
300.0848.
Acknowledgements
We thank DST, Government of India, for financial support
and the Sophisticated Analytical Instrumentation Facility
(SAIF); IITM for the NMR spectra.
References and notes
1. Strauss, U. T.; Faber, K. Tetrahedron: Asymmetry 1999, 10,
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4.4.8. (3E,5E)-Methyl-2-hydroxy-6-(o-nitrophenyl)hexa-
3,5-dienoate (8h). Yellow liquid; 1H NMR (400 MHz,
CDCl3) d: 1.53 (br s, 1H), 3.86 (s, 3H), 4.70 (d, J¼5.5 Hz,
1H), 5.89 (dd, J¼15.1, 5.5 Hz, 1H), 6.57 (dd, J¼15.1,
10.6 Hz, 1H), 6.69 (dd, J¼15.1, 10.6 Hz, 1H), 6.99 (d,
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1H); 13C NMR (400 MHz, CDCl3) d: 52.4, 72.1, 124.5,
127.9, 128.2, 129.3, 130.4, 130.7, 132.0, 132.8, 137.9,
146.7, 181.9; IR nmax (CHCl3): 3672, 1732, 1685, 1396,
1005, 962, 790 cmꢁ1; HRMS (ESI): found 286.0691,
C13H13NO5Na [M+Na]+ requires 286.0691.
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1H), 4.25 (q, J¼6.9 Hz, 2H), 4.79 (d, 1H, J¼5.5 Hz), 6.70
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1H), 7.04–7.19 (m, 4H); 13C NMR (400 MHz, CDCl3)
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129.5, 129.7, 130.4, 133.4, 134.4, 173.0; IR nmax (KBr):
3444, 2982, 2361, 2093, 1732, 1667, 1607, 1472, 1441,
1371, 1130, 1084, 978, 860, 676, 587, 466 cmꢁ1; HRMS
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(400 MHz, CDCl3) d: 1.29 (d, J¼11.2 Hz, 6H), 3.07 (br s,
1H), 4.70 (d, J¼5.3 Hz, 1H), 5.11 (sep, J¼11.1 Hz, 1H),
5.84 (dd, J¼15.6, 5.3 Hz, 1H), 6.59 (dd, J¼15.6, 10.7 Hz,
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2982, 2936, 2556, 1730, 1454, 1375, 1278, 906, 862, 755,
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C15H19O3 [M+H]+ requires 247.1334.

V. Thangavel, A. Chadha / Tetrahedron 63 (2007) 4126–4133
4133
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