Asymmetric reduction of alkyl 2-oxo-4-arylbutanoates and -but-3-enoates by Candida parapsilosis ATCC 7330: Assignment of the absolute configuration of ethyl 2-hydroxy-4-(p-methylphenyl)but-3-enoate by 1H NMR

Article abstract of DOI:10.1016/j.tetasy.2004.11.002

Enantioselective bioreduction of alkyl 2-oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates mediated by Candida parapsilosis ATCC 7330 resulted in the formation of the corresponding (S)-2-hydroxy compounds in high enantiomeric excesses (93-99%) and good isolated yields (58-71%). The absolute configuration of enantiomerically pure ethyl 2-hydroxy-4-(p-methylphenyl)but-3-enoate obtained by the reduction of the corresponding keto ester was assigned by ¹H NMR using Mosher's method.

Full text of DOI:10.1016/j.tetasy.2004.11.002

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3961–3966
Asymmetric reduction of alkyl 2-oxo-4-arylbutanoates and
-but-3-enoates by Candida parapsilosis ATCC 7330:
assignment of the absolute configuration of ethyl
1
2-hydroxy-4-(p-methylphenyl)but-3-enoate by H NMR
Baburaj Baskar, N. Ganesh Pandian, Kuttikode Priya and Anju Chadha*
Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
Received 12 October 2004; accepted 1 November 2004
Abstract—Enantioselective bioreduction of alkyl 2-oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates mediated by Candida para-
psilosis ATCC 7330resulted in the formation of the corresponding ( S)-2-hydroxy compounds in high enantiomeric excesses (93–
99%) and good isolated yields (58–71%). The absolute configuration of enantiomerically pure ethyl 2-hydroxy-4-(p-methylphen-
1
yl)but-3-enoate obtained by the reduction of the corresponding keto ester was assigned by H NMR using MosherÕs method.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
aromatic and aliphatic ketones.15 The involvement of
oxidoreductases in deracemisation reactions via asym-
metric reduction is also well explained.16,17 Recently,
we reported the deracemisation of racemic a- and b-
hydroxy esters to the corresponding enantiomerically
pure (S)-hydroxy esters in 62–85% chemical yields and
15–99% ees using whole cells of C. parapsilosis ATCC
7330.17a,b Herein we report for the first time the asym-
metric reduction of a-oxo esters, specifically alkyl 2-
oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates
using the whole cells of C. parapsilosis ATTC 7330into
their corresponding 2-hydroxy esters in high conversion
and high ee.
Enantiomerically pure alkyl 2-hydroxy-4-phenylbutano-
ates are important chiral building blocks,1 particularly
the b,c-unsaturated a-hydroxy esters, as they can also
lend themselves to stereoselective epoxidations, such as
Sharpless epoxidation,2 dihydroxylation3 and amino
hydroxylation.4 These chiral molecules can be prepared
by both chemical5 and biocatalytic6,7 methods. Enzy-
matic resolution,7 a widely used method, results in
50% of the unwanted enantiomer unlike biocatalytic
asymmetric reduction, which ideally gives only one
enantiomer in high enantiomeric excess and chemical
yield.8 Isolated oxidoreductases, coupled with cofactors
are known to reduce 2-oxo esters to enantiomerically
pure 2-hydroxy esters,9 but these enzymes in whole cells
have the advantage of regenerating the cofactors viz
NAD(P)H within the cell.10 Among the whole cells,
plant tissue culture,11 bacteria12 and yeast13,14 have been
used. These reactions either require too much time (10
days for D. carota) or result in lower chemical yields
and enantiomeric excesses as seen in bakerÕs yeast13
and Pseudonocardia thermophila IFO 12133.12 Purified
carbonyl reductases from Candida parapsilosis DSM
70125 give highly stereoselective reductions of various
2. Results and discussion
2.1. Reduction of methyl and ethyl esters of 2-oxo-4-aryl-
butanoic acid
The enantioselective reduction of ethyl and methyl esters
of 2-oxo-4-arylbutanoic acids 1a–f and 2-oxo-4-arylbut-
3-enoic acids 2a–f using whole cells of C. parapsilosis
ATCC 7330provided the respective ethyl and methyl
esters of (S)-2-hydroxy-4-arylbutanoic acids 3a–f (Scheme
1A) and (S)-2-hydroxy-4-arylbut-3-enoic acids 4a–f
(Scheme 1B) in excellent enantiomeric excess (93–99%)
and high isolated yields (58–71%). At the end of the
reaction (i.e., after complete consumption of the a-keto
*
Corresponding author. Tel.: +91 44 2257 8258; fax: +91 44 2257
8241; e-mail: anjuc@iitm.ac.in
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.002

3962
B. Baskar et al. / Tetrahedron: Asymmetry 15 (2004) 3961–3966
OH
O
This reaction involves the preincubation of bakerÕs yeast
(A)
Candida parapsilosis
O
O
with the additive phenacyl chloride and the enantio-
meric excesses (48–99%) and chemical yields (18–99%)
depending on the concentration of the additive.13a We
have previously reported the asymmetric reduction of
1b and 2a–f using plant tissue culture cells of Daucus
carota,11,19 which resulted in the formation of the (R)-
hydroxy ester. Herein we report the use of C. parapsilo-
sis ATCC 7330for biocatalytic asymmetric reduction of
these compounds to give the enantiomerically pure (S)-
2-hydroxy esters indicating different enantiospecificities
of enzymes from different sources. C. parapsilosis ATCC
7330mediated reduction of a-keto esters and their p-
substituted derivatives 1a–f and 2a–f gave the corre-
sponding a-hydroxy esters in good isolated yields
(58–69%) and high ees (93–96%) (Table 1) regardless
of the electronic properties of the substituents suggesting
the wide range of substrates the enzyme can accept. In
addition to high enantioselectivity, the biocatalyst dis-
plays chemoselectivity in that C. parapsilosis ATCC
7330mediated reduction of C @O precedes that of
C@C for compounds 2a–f. Hence, unsaturated a-hydr-
oxy esters, 4a–f are obtained after 4h, while saturated
alcohols 3a–f appear after 6–10h. Thus, both the
unsaturated hydroxy ester and the saturated hydroxy
esters can be obtained by merely altering the time of
the reaction. Notably, the chemical reduction of ethyl
2-oxo-4-phenylbut-3-enoate using sodium borohydride
resulted in the formation of unsaturated diol while
palladium/carbon gave the corresponding saturated
a-hydroxy ester.22
R'
R'
R'
4hr, 25oc
O
O
R
R
R
(S)-3a-3f
Keto ester- 1a-1f
Yield = 60-71%,
ee = 90-99%
OH
O
(B)
Candida parapsilosis
O
O
R'
4hr, 25oc
O
O
R
(S) -4a-4f
Keto ester- 2a-2f
Yield =52-65%,
ee = 91-98%
1- 4
a. R = H;
b. R = H;
R' = CH3
R' = CH2CH3
c. R = p-Cl; R' = CH3
d. R = p-Cl; R' = CH2CH3
e. R = p-Me; R' = CH3
f. R = p-Me; R' = CH2CH3
Scheme 1. Asymmetric reduction by C. parapsilosis ATCC 7330of (A)
alkyl 2-oxo-4-arylbutanoates 1a–f; (B) alkyl 2-oxo-4-arylbut-3-enoates
2a–f.
esters), the cells were filtered and the aqueous layer ex-
tracted with ethyl acetate. The enantiomeric excesses
of the product 2-hydroxy esters were determined by chi-
ral HPLC analysis. Chiral chemical catalysts for the
preparation of 2a,5a 2b5b and 4a5d have also been re-
ported. Resolution of methyl 2-hydroxy-4-phenylbut-3-
enoate 2a using Pseudomonas sp. lipase7b resulted in
the (R)-alcohol in 49% chemical yield and 88% ee while
the (S)-acetate was obtained in 43% chemical yield and
98% ee. Asymmetric reduction of a-keto acids of esters
2a–d using Proteus vulgaris18 gave the corresponding
enantiomerically pure a-hydroxy acids in 85–95% chem-
ical yields and >97% ees. However, the reaction requires
stringent anaerobic conditions. Preparation of enantio-
merically pure (R)-alkyl and aryl 2-hydroxy-4-phenyl-
butanoates by bakerÕs yeast mediated asymmetric
reduction of the corresponding 2-oxo esters resulted in
yields ranging from 80% to 90% and ees of >90%.13b
An important consideration for a catalytic reaction is
the reusability of the catalyst. In order to increase the
robustness of whole cell biocatalysts they are often
encapsulated in gels,8b which allows for easy separation
of the biocatalyst and simplifies the downstream
processing to separate the product. Even though algi-
nated cells used in this study resulted in a slightly less
yield over free cells, the same could be recovered and re-
used up to three cycles without loss in activity (Table 1).
Table 1. Microbial reduction of ethyl and methyl esters of 2-oxo-4-arylbutanoic acids 1a–f and 2-oxo-4-arylbut-3-enoic acids 2a–f using whole cells
of C. parapsilosis ATCC 7330
Entry
Free cells of C. parapsilosis ATCC 7330Entry
Immobilised cells of
Abs. config.
C. parapsilosis
Yield (%)
Ee (%)
[a]D Values
Ref.
Yield (%)
Ee (%)
Abs. config
3a
3b
3c
3d
3e
3f
68
71
65
67
68
69
64
63
58
59
65
62
99
99
95
94
93
93
98
98
95
96
95
94
+32.2 (c 2.1, CHCl3)
+21.4 (c 1, CHCl3)
+22.3 (c 0.57, CHCl3)
+17.5 (c 1, CHCl3)
+28.7 (c 1.05, CHCl3)
+18.1 (c 0.6, CHCl3)
+67 (c 1, CHCl3)
+21.4 (c 1.1, CHCl3)a
+55.5 (c 1.25, CHCl3)
+18.8 (c 1, CHCl3)a
+50.7 (c 1.1, CHCl3)
+40.9 (c 1.2, CHCl3)
20
13a
18
S
3a
3b
3c
3d
3e
3f
63
65
97
98
S
S
Sb
S
6096
64
S
13a
—
S
93
91
90
96
97
96
91
Nd
93
S
Nd
Sc
S
62
64
Nd
S
—
4a
4b
4c
4d
4e
4f
21
4a
4b
4c
4d
4e
4f
54
55
S
13a
18
S
Sb
S
52
54
S
13a
—
S
S
Nd
Sc
6096
61
—
S
a Specific rotation was taken after hydrogenation.
b Absolute configuration was assigned after hydrolysis.
c Absolute configuration was assigned by MosherÕs method. Nd = not determined.

B. Baskar et al. / Tetrahedron: Asymmetry 15 (2004) 3961–3966
3963
2.2. Role of the solvent in cofactor regeneration
phenyl ring of MosherÕs ester as shown in Figure 1a
and b. However, the diamagnetic effect of the phenyl
ring on the protons of the –OCH2CH3 group in
MTPA-(RR)-4b and MTPA-(SR)-4b is less as seen from
the difference in the d values (dS–R = <0.03ppm and
–OCH2, dS–R = <0.02ppm) due to a larger distance be-
tween the –OCH2CH3 moiety and the phenyl ring.
Experiments done with ethanol and isopropanol re-
sulted in complete conversion of the a-keto esters 1a–f
and 2a–f into a-hydroxy esters 3a–f and 4a–f. This could
be due to the regeneration of cofactors (NAD+ or
NADP+)23 by alcohol while in nonalcoholic solvents,
such as hexane, tetrahydrofuran, dimethylformamide,
benzene, dimethyl sulfoxide and chloroform, the reac-
tion did not go to completion.
(b) An exact match in d-values for protons Ha and Hb
was seen in individual diastereomers MTPA-(RR)-4b
and MTPA-(SR)-4b and a mixture of diastereomers
[MTPA-(R,R and S,R)-4b] (Fig. 2). Therefore, the
2.3. Assignment of the absolute configuration of 4f
The absolute configuration of compounds 3a,b,d and 4a
was confirmed to be ÔSÕ by comparing their specific rota-
tions with those reported.13a,20,21 Dao et al. have as-
signed the absolute configuration of ethyl (p-methyl
phenyl)butanoates 3f13a by comparing the retention
times of the corresponding unsubstituted, p-methoxy
and m-chloro esters on chiral HPLC. However, a mere
comparison of retention times is not a confirmation of
absolute configuration. Herein, the absolute configura-
more shielded
O
Ha
MeO
O
MeO
O
Ph
CF3
Ph
CF3
H3C
Hb
O
Hb
H3C
O
O
O
H
Ha
H
O
less shielded
MTPA-(S,R)-4b (b)
MTPA-(R,R)-4b (a)
1
tion of 4f was determined by a detailed H NMR study
of the MosherÕs derivative24 of 4f. (R)-MTPA esters of
(RS)-4b, (R)-4b, (S)-4b were used as a reference for this
study. (R)-MTPA esters of (RS)-4f and enantiomerically
pure unknown 4f were also prepared. A typical reaction
is shown in Scheme 2. Enantiomerically pure (R)-4b was
prepared according to the reported literature.19 The
structure of the diastereomers was confirmed by 1H
NMR spectroscopy as per the following discussion:
Figure 1. (R)-MTPA derivatives of (R,R)-4b and (S,R)-4b.
(a) Figure 1a and b represents the absolute configura-
tions of diastereomers of MTPA-(R,R)-4b and MTPA-
(S,R)-4b obtained from (S)-MosherÕs acid chloride with
(R)-4b and (S)-4b, respectively. Hence, the arrangement
of the phenyl ring present in MosherÕs acid moiety can
be assigned as either cis or trans to the olefinic protons
of (R)-4b or (S)-4b. The proton resonating upfield is
due to the diamagnetic effect of phenyl ring present in
the MosherÕs acid.25 In the ester MTPA-(R,R)-4b pre-
pared from (R)-4b and (S)-MTPA Cl, the Ha
(d = 6.21–6.27ppm) and Hb protons (d = 6.67–
6.71ppm) are more shielded as compared to the Ha
(d = 6.28–6.34ppm) and Hb (d = 6.8–6.84ppm) in
MTPA-(SR)-4b, which is derived from (S)-4b and (S)-
MTPA Cl. This is due to the diamagnetic effect of the
Figure 2. Signals of Ha and Hb in MTPA-(R,R and S,R)-4b, MTPA-
(S,R)-4b, MTPA-(R,R)-4b and MTPA-4f.
MeO Ph
MeO
O
Ph
CF3
O
Cl
CF3
O
(S)
H3C
O
O
Pyridine, rt, 24h
H
H
+
OH
O
CH2CH3
O
R
R
(R,R or S,R or R,R and S,R)-MTPA esters
(R or S or RS)
R=H, MTPA-(RR and SR)-4b
R=H, MTPA-(RR)-4b
R=H, MTPA-(SR)-4b
R=Me, MTPA-(RR and SR)-4f
R=Me, MTPA-4f
R= H, (RS)-4b
R= H, (R)-4b
R= H, (S)-4b
R= Me, (RS)-4f
R= Me, 4f
Scheme 2. Synthesis of (R)-MTPA esters of (R,S)-4b, (R)-4b, (S)-4b, (RS)-4f and 4f.

3964
B. Baskar et al. / Tetrahedron: Asymmetry 15 (2004) 3961–3966
H3C
tion conditions. The method is better than all the re-
more shielded
ported methods so far, as the biocatalyst shows high
chemo-, and enantioselectivity towards b,c-unsaturated
and saturated a-hydroxy esters. With appropriate
immobilisation techniques, this method can be suitably
scaled up.
O
Ha
MeO
O
MeO
Ph
CF3
Ph
CF3
H3C
Hb
O
O
Hb
H3C
O
O
O
H
Ha
H
O
less shielded
(S,R)- MTPA (b)
(R,R)- MTPA (a)
H3C
4. Experimental
Figure 3. (R)-MTPA derivatives of (R,R)-4f and (S,R)-4f.
4.1. General methods
known (R)-4b and (S)-4b-ethyl 2-hydroxy-4-phenylbut-
3-enoates were reconfirmed by H NMR spectroscopy
1
1H and 13C NMR spectra were recorded in CDCl3 solu-
tion on a Bruker AV-400 spectrometer operating at 400
and 100MHz, respectively. Chemical shifts are ex-
pressed in ppm values using TMS as an internal stand-
ard. HPLC analysis was done on a Jasco PU-1580
liquid chromatograph equipped with a manual injector
(20lL) and a PDA detector. The columns used
were Chiralcel OD-H and Chiralcel OJ-H (Daicel,
4.6 · 250mm). The enantiomeric excesses (% ee) of 3a–f
and 4a–f were determined by HPLC analysis. The eluent
used was hexane/isopropanol (98:2) at a flow rate of
1mLminÀ1 and the absorbance monitored using a
PDA detector at 254nm. Optical rotations were deter-
mined on a Jasco Dip 370digital polarimeter. TLC
was done on Kieselger 60F 254 aluminium sheets (Merck
1.05554). All starting materials were prepared according
to literature procedures.27
using MosherÕs method. Having assigned the absolute
configuration of known enantiomers of (R)-4b and (S)-
4b-ethyl 2-hydroxy-4-phenylbut-3-enoates, this method
was employed to establish the absolute configuration
of the optically pure unknown enantiomer 4f. The only
structural difference between known enantiomers (R)-4b
and (S)-4b and unknown enantiomer 4f was the methyl
substituent at the para position on the aromatic ring,
which was assumed to have no effect on the protons
under consideration for this study.
1
(c) The H NMR spectrum of MTPA-4f (Ha = 6.22–
6.28ppm and Hb = 6.76–6.80ppm) was compared
with the H NMR spectrum of the mixture of MTPA-
1
(R,R and S,R)-4f (Ha = 6.22–6.28 and 6.15–6.21ppm,
Hb = 6.76–6.80and 6.64–6.68). These d-values for Ha and
Hb protons match with the MTPA-SR (less shielded)
as shown in Figure 3 indicating that the unknown opti-
cally pure enantiomer 4f is ÔS Õ.
4.2. Culture medium of the microorganism
The yeast, C. parapsilosis (ATCC 7330) was grown in
YMB medium (60mL) in 250mL Erlenmeyer flasks.
The flasks were incubated at 25ꢁC with a shaking speed
of 200rpm for 40h. The cells were pelleted down by cen-
trifugation at 3214g for 15min, washed with distilled
water and used for the biotransformation reaction.
(d) The splitting pattern in the 1H NMR spectra of
MTPA-(R,R and S,R)-4f was similar to that obtained
with the MTPA-(R,R and S,R)-4b and splitting pattern
of MTPA-4f was similar to that obtained with MTPA-
(S,R)-4b thus corroborating the (S)-configuration (Fig.
2). The absolute configuration of compound 3f was
determined by comparing the retention time on chiral
HPLC of the corresponding saturated compound ob-
tained after hydrogenating the unsaturated compound
4f. In the case of the unsaturated compounds 4b and
4d, the absolute configuration was determined by hydro-
genating and comparing them with 3b and 3d, respec-
tively (Chiral HPLC). All these were found to be ÔS Õ.
The specific rotation of the a-hydroxy acid obtained
after the hydrolysis of ester 4c was measured and com-
pared with that reported in literature18 based on which
the absolute configuration was assigned as ÔS Õ. Thus,
the reductase of C. parapsilosis ATCC 7330, which is
known to play an important role in deracemisation of
a-hydroxy esters26 can be safely called the (S)-specific
reductase for a-oxo esters.
4.3. Determination of conversion and enantiomeric excess
of the product
Keto ester 1b (0.39mmol, 80mg) dissolved in ethanol
(2mL) was added into an Erlenmeyer flask containing
the cells (20g wet wt) in water (40mL) and stirred on
an orbital shaker at 150rpm for 4h at 25 ꢁC. The conver-
sions were determined by HPLC using a Sil-C18 column
(size 4.6 · 250mm) by comparing the retention time of
the product obtained with that of the keto ester (reac-
tant) and standard hydroxy ester (expected product).
The eluent used was acetonitrile and water (85:15).
The absence of keto ester 1b by HPLC was taken to
indicate that all the keto ester was consumed at which
point the contents of the flask were transferred into a
centrifuge tube. The broth was centrifuged (3214g for
30min) and the supernatant decanted and filtered. The
filtrate (40mL) was extracted with ethyl acetate
(2 · 20mL), the organic phase separated, dried over
anhydrous Na2SO4, filtered and concentrated. The
crude product was purified by chromatography on silica
gel (mesh size 100–200) using 5% ethyl acetate–hexane,
to give 3b as colourless oil (0.313mmol, 65mg, 71%).
The ees of the hydroxy esters 3a–f and 4a–f were deter-
3. Conclusion
We have established a convenient and simple procedure
for the enantioselective reduction of alkyl 2-oxo-4-aryl-
butanoates 1a–f and 2-oxo-4-phenylbut-3-enoates 2a–f
using whole cells of C. parapsilosis ATCC 7330to give
the (S)-2-hydroxy ester in good yields (58–71%) and
high enantiomeric excesses (93–99%) under mild reac-

B. Baskar et al. / Tetrahedron: Asymmetry 15 (2004) 3961–3966
3965
mined by chiral HPLC using Chiralcel OD-H and OJ-H
column, respectively, using hexane/isopropanol (98:2) as
the mobile phase. The other keto esters, 1a,c–f and 2a–f
were also reduced by employing the same procedure and
afforded the (S)-a-hydroxy ester in excellent ee (93–
99%). The 1H and 13C NMR (400 and 100MHz)
of the methyl and ethyl esters of 2-hydroxy-4-arylbutanoic
acid and 2-hydroxy-4-arylbut-3-enoic acids 3a,b,d,f and
4a–f were identical to those reported earlier.13a,18–20
(iv) MTPA-(R,R and S,R)-4f: 1H NMR (400MHz,
CDCl3): 1.23–1.32 (2t, 6H, 2 · –OCH2CH3),
2.33 (s, 3H, –CH3Ph), 2.34 (s, 3H, –CH3Ph),
3.59 (s, 3H, –OCH3), 3.696 (s, 3H, –OCH3)
4.22–4.30(2q, 4H, 2 · –OCH2CH3), 5.78–5.789
(2d, 2H, 2 · –CH3 Ph–CH@CH–CH), 6.15–
6.21 (dd, 1H, –CH3Ph–CH@CH–CH), 6.22–6.28
(dd, 1H, –CH3Ph–CH@CH–CH), 6.64–6.68 (d,
1H, –CH3Ph–CH@CH–CH), 6.76–6.80(d, 1H,
–CH3Ph–CH @CH–CH) and 7.13–7.66 (m, 18H,
2 · CH3C6H4–, 2 · C6H5–).
4.4. Asymmetric reduction of a-keto esters using immo-
bilised cells of C. parapsilosis ATCC 7330
(v) MTPA-(S,R)-4f: 1H NMR (400MHz, CDCl3):
1.23–1.27 (t, 3H, –OCH2CH3), 2.34 (s, 3H,
–CH3Ph), 3.59 (s, 3H, –OCH3), 4.22–4.28 (q, 2H,
OCH2CH3), 5.76–5.78 (2d, 2H, –CH3 Ph–CH@CH–CH),
6.22–6.28 (dd, 1H, –CH3Ph–CH@CH–CH), 6.76–
6.80(d, 1H, –CH 3Ph–CH@CH–CH) and 7.13–
7.66 (m, 9H, CH3C6H4–, C6H5–).
Keto ester 1b (0.39mmol, 80mg) dissolved in ethanol
(2mL) was reduced using alginated beads of C. parapsi-
losis ATCC 7330(20g, wet weight) in 40mL of water.
The reaction mixture was incubated at 25ꢁC for 4h with
a shaking speed of 150rpm. The crude product was puri-
fied as mentioned for free cells to give 3b as a colourless
oil (0.25mmol, 52mg, 65%). Although the (S)-alcohol
was formed in all the reductions using alginated cells,
the ee and yield of the product was slightly lower than
that obtained using free cells. The recovered cells were
reused for up to three cycles without any loss in activity.
Acknowledgements
We thank the Department of Science and Technology,
Government of India for funding this research.
4.5. Preparation of MTPA-(R,R and S,R)-4b
References
To a solution of racemic ethyl 2-hydroxy-4-phenylbut-3-
enoate (RS)-4b (10mg, 0.048mmol) in pyridine (100lL)
was added (S)-MTPA chloride (20mg, 0.0794mmol)
and the solution allowed to stand at room temperature
for 24h. The reaction mixture was then washed with
dil HCl, followed by aqueous Na2CO3. The product
was extracted using ethyl acetate (4 · 1mL) and concen-
trated to obtain the crude product, which was purified
by preparative TLC using hexane and ethyl acetate
(95:5) as mobile phase eluent. The product was charac-
terised by the 1H NMR spectroscopy. The MTPA esters
of (R)-4b, (S)-4b, (RS)-4f and 4f were also prepared by
this procedure (Scheme 2).
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