Diastereo- and enantioselective bioreduction of ethyl 2-(4-chlorophenoxy)- 3-oxobutanoate clofibrate analogues by Kluyveromyces marxianus and other whole cell biocatalysts

Article abstract of DOI:10.1016/j.tetasy.2004.08.028

Growing and resting cells of several yeasts, which catalyze the hydride transfer to a carbonyl, were screened and used in conditions to find out the suitable methodology to prepare clofibrate analogues. Clofibrate is an antilipidemic drug. In particular, the bioreduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1 was investigated to separately prepare the four possible stereoisomers of the ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate 2. Compound (2R,3S)-2 was prepared with ee = 97% and 73% yield in the presence of Kluyveromyces marxianus; (2S,3S)-2 preparation with ee >99% in 9% and 33% yield was mediated by Saccharomyces cerevisiae CBS 7336 and Trigonopsis variabilis, respectively. Diastereomeric excess values of all the reactions investigated were up to >99%. Furthermore, enantiomeric excesses of the bioconversions varied between 2% and >99% using growing cells and, 12% and >99% using resting cells. The absolute configuration of (2R,3S)-2 was established by X-ray analysis of the corresponding acid 3.

Full text of DOI:10.1016/j.tetasy.2004.08.028

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3511–3517
Diastereo- and enantioselective bioreduction of ethyl
-(4-chlorophenoxy)-3-oxobutanoate clofibrate analogues by
2
Kluyveromyces marxianus and other whole cell biocatalysts
a
Maria Grazia Perrone, Ernesto Santandrea, Antonio Scilimati, Christoph Syldatk,
a
a,
b
*
a
Vincenzo Tortorella, Francesco Capitelli and Valerio Bertolasi
c
d
a
Dipartimento Farmaco-Chimico, Universit a` di Bari, Via E.Orabona 4, 70125 Bari, Italy
b
Universit a¨ t Karlsruhe (TH), Engler-Bunte-Institut, Lehrstuhl f u¨ r Technische Biologie,
Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany
c
Istituto di Cristallografia (IC-CNR), Via Amendola 122/o, 70125 Bari, Italy
d
Dipartimento di Chimica and Centro di Strutturistica Diffrattometrica,
Universit a` di Ferrara, Via Borsari 46, 44100 Ferrara, Italy
Received 29 July 2004; accepted 20 August 2004
Abstract—Growing and resting cells of several yeasts, which catalyze the hydride transfer to a carbonyl, were screened and used in
conditions to find out the suitable methodology to prepare clofibrate analogues. Clofibrate is an antilipidemic drug. In particular, the
bioreduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1 was investigated to separately prepare the four possible stereoisomers of
the ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate 2. Compound (2R,3S)-2 was prepared with ee = 97% and 73% yield in the pres-
ence of Kluyveromyces marxianus; (2S,3S)-2 preparation with ee >99% in 9% and 33% yield was mediated by Saccharo-
myces cerevisiae CBS 7336 and Trigonopsis variabilis, respectively. Diastereomeric excess values of all the reactions investigated were
up to >99%. Furthermore, enantiomeric excesses of the bioconversions varied between 2% and >99% using growing cells and, 12%
and >99% using resting cells. The absolute configuration of (2R,3S)-2 was established by X-ray analysis of the corresponding acid 3.
ꢀ
2004 Elsevier Ltd. All rights reserved.
1
. Introduction
such a hydride transfer with very high regio- and stereo-
1
2
selectivity.
usually consist of mild reaction conditions and make
Furthermore, biochemical approaches
Chiral alcohols are very important intermediates in
organic synthesis and precursors of a large number of
2
2
use of environmentally safe reagents. An increasing
number of microorganisms have shown the ability of
mediating the reduction reaction of the carbonyl with
1
–5
pharmaceuticals.
One of the easiest and widely used
synthetic routes to prepare this crucial class of com-
pounds is the chemical and chemo-enzymatic asymmet-
ric reduction of prochiral ketones. Various chiral
2
3
high stereoselectivity.
6
–9
reducing agents
aluminium-hydrides for hydride transfer,
reduction with chiral transition metal complexes
or chirally modified boron- and
Clofibrate [ethyl 2-(4-chlorophenoxy)-2-methylpropano-
ate], a well-known lipid-modifying agent, has been
proven to be active in the treatment of human dyslipa-
1
0,11
catalytic
1
2–18
2
4–28
are the most used chemical methods to enantioselec-
tively reduce the carbonyl. Chemo-enzymatic hydride
transfer represents a valuable alternative to chemical
The success of such biotransforma-
tions is mainly due to the presence in the cells of
enzymes, involved in the reaction, capable of mediating
emias
induced cardiac hypertrophy. Nevertheless, it shows,
and more recently in rat pressure overload
2
9
especially in humans, a number of adverse side
30–33
1
9,20
methodologies.
effects.
analogues with an improved pharmacological pro-
Hence, the development of new clofibrate
3
4–36
file,
to efficaciously cure the hyperlipidaemias and
other cardiovascular diseases is crucial. A new series of
chiral clofibrate analogues has been prepared by us as
racemates and has already been tested. Unfortunately,
they were inactive in a transactivation assay aimed at
*
Corresponding author. Tel.: +39 080 5442762; fax: +39 080
442231; e-mail: ascilimati@farmchim.uniba.it
5
0
957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.08.028

3512
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3511–3517
O
COOEt
O
COOEt
Cl
HO
Cl
HO
anti-(2R,3R)-2
syn-(2S,3R)-2
O
O
COOEt
[
H]
Cl
O
COOEt
O
HO
COOEt
1
Cl
HO
Cl
syn-(2R,3S)-2
anti-(2S,3S)-2
Figure 1. The stereoisomers of ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate 2 obtainable by the reduction of ethyl 2-(4-chlorophenoxy)-3-
oxobutanoate 1.
evaluating their capacity to interact with peroxisome
3
Kluyveromyces marxianus CBS 6556, Saccharomyces
cerevisiae DSM 11285, Saccharomyces cerevisiae CBS
7336, Cryptococcus curvatus ATCC 29509, Candida
bombicola ATCC 22214, Trigonopsis variabilis DSM
70714 were then selected for scaling up the reduction
of 1. The reactions were performed by using both grow-
ing and resting cells. The results obtained are reported in
Tables 1 and 2, respectively.
7
proliferator-activated receptor isoform a (PPARa).
Thus, single stereoisomers were needed for the bioassay,
to check the hypothesis that the lack of activity was not
due to molecular structural features, but due to a crowd-
ing in the ligand binding domain of the receptor, as the
racemate of each compound was assayed.
The main challenge in the preparation of 2, in an enan-
tiomerically pure form, is represented by the presence of
two stereogenic centres in the molecule, involving the
possible formation of four stereoisomers (Fig. 1).
With growing cells, all of the strains showed moderate
to high diastereo- and enantioselectivity (Table 1).
Notable conversions were obtained for T. variabilis
(80%), S. cerevisiae DSM (89%), C. bombicola (91%)
Growing and resting cells of different yeast strains have
been used, as a tool for the diastereo- and enantioselec-
tive reduction of ethyl 2-(4-chlorophenoxy)-3-oxobuta-
noate 1 and with the purpose of finding a procedure
to possibly obtain separately the four stereoisomers of
and for K. marxianus (quantitative), whereas it was lower
(59%) for S. cerevisiae CBS. When Cr. curvatus was
employed, the extent of conversion was only 14%.
In all cases, the main pair of stereoisomers turned out to
be the syn form, with diastereomeric excesses ranging
from 32% (T. variabilis and Cr. curvatus) to >99%
2
in high chemical yield and high enantiomeric excess.
(
K. marxianus). The absolute configuration of 2 formed
in the bioreduction performed in the presence of
K. marxianus was established to be (2R,3S) by single
crystal X-ray analysis of the corresponding acid 3 (quan-
titatively obtained by treating 2 with KOH) (Fig. 2). All
the other absolute configurations were assessed by
comparing the HPLC retention times of standard com-
2
. Results and discussion
BakerÕs yeast is commonly employed in bioreduction
reactions because of its simplicity of use. Thus, as a first
attempt, commercially available BakerÕs yeast was used
by us. In this case, we observed a quantitative conver-
sion of the substrate (c = 5g/L) into the product with
very high diastereomeric and enantiomeric excess
3
8
39
pounds prepared from threonines. (2R,3S)-2 was the
most abundant enantiomer within the syn pair formed
in the presence of all the microorganisms listed in Table
1. EeÕs varied from 42% with T. variabilis to 92% with S.
cerevisiae DSM 11285. In the case of S. cerevisiae CBS
7336, almost racemic syn-2 (ee = 2%) was obtained.
Within the anti pair, (2S,3S)-2 was the only enantiomer
formed in the reduction mediated by S. cerevisiae CBS
7336 and T. variabilis (>99%), even if in low yields
(9% and 33%, respectively). Ee = 42% for C. bombicola.
(
de = 92%, eesyn pair = 94%). However, in principle look-
ing for better results and eventually reverse stereoselec-
tivity, many other microorganisms may be used to
induce the same transformation. Next, different yeast
strains were screened (Scheme 1) to improve the process
mediated by BakerÕs yeast, and to possibly obtain sepa-
rately the four stereoisomers of 2.
O
O
COOEt
O
COOEt
*
growing or resting
*
whole cell biocatalyst
Cl
Cl
HO
1
2
Scheme 1. Bioreduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1.

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3511–3517
3513
Table 1. Reduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1 with growing cells of different microorganisms
a
Yield (%)
b
Conv. (%)
c
De (%)
d
d
20
½aŠD
Microorganism
t
R
(h)
Eesyn (%)
Eeanti (%)
S. cerevisiae DSM
S. cerevisiae CBS
C. bombicola
8
61
40
53
79
5
89
59
81
83
92
2
66
>99
42
+27.6
+18.8
+24.3
+24.4
+0.8
24
24
29
25
8
91
80
56
32
72
42
T. variabilis
>99
e
ND
––
e
Cr. curvatus
14
Quantitative
32
>99
ND
86
K. marxianus
73
+31.3
a
b
c
Yields refer to the product isolated by chromatography.
1
Conversion = percentage of conversion ( H NMRdata).
1
Diastereomeric excesses (syn > anti pair) determined by H NMR .
39
d
e
Enantiomeric excesses were determined by HPLC.
ND = not determined due to low conversion.
Table 2. Reduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1 with resting cells of different microorganisms
a
Yield (%)
b
Conv. (%)
c
De (%)
d
d
20
½aŠD
Microorganism
t
R
(h)
Eesyn (%)
Eeanti (%)
S. cerevisiae DSM
S. cerevisiae CBS
C. bombicola
3.5
2
79
41
53
86
31
84
Quant.
95
95
84
87
85
63
93
98
98
97
24
>99
66
+51.0
+13.3
+12.5
+13.3
+5.6
5
54
27
T. variabilis
2.5
4
94
43
55
12
Cr. curvatus
K. marxianus
11
>99
1.5
Quant.
––
+48.9
a
b
c
Yields refer to the product isolated by chromatography.
1
Conversion = percentage of conversion ( H NMRdata).
1
Diastereomeric excesses (syn > anti form) determined by H NMR.
39
d
Enantiomeric excesses were determined by HPLC.
Figure 2. ORTEP view of the asymmetric unit with the atomic numbering scheme of (2R,3S)-2-(4-chlorophenoxy)-3-hydroxybutanoic acid 3.
Thermal ellipsoids probability level at 30%.
It is noteworthy that the enantioselectivity and enzy-
matic activity (different reaction times) depended on
the strain involved in the biotransformation, while the
use of a different strain of the same microorganism, as
in the case of S. cerevisiae, led to quite different results
in terms of conversion (89% and 59%, respectively).
These differences can be ascribed to the variable extent
of expression of the enzymes involved in the
vity and a high enantioselectivity was observed, the
(2R,3S) being the major stereoisomer formed. This re-
sult is interesting if compared to a recently reported
reduction of b-keto ester precursor of b-lactam group
antibiotics, in which a (3R)-3-hydroxy compound was
formed by reducing the carbonyl group in the presence
of K. marxianus KCTC 7155. Furthermore, by consid-
ering the reaction times, K. marxianus shows the highest
reducing activity together with S. cerevisiae DSM 11285.
4
3
2
1
bioreduction.
The best results were obtained when using resting cells
of K. marxianus, which have proven to be useful in bio-
Biotransformations performed using resting cells were
faster than the corresponding reactions accomplished
with growing cells (Table 2). Diastereoselectivity was
4
0–42
reductions.
In this case, a complete diastereoselecti-

3514
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3511–3517
almost similar under the two conditions, with the excep-
tion of Cr. curvatus, for which a lower de value (11%
with resting cells and 34% with growing cells) was
obtained. Enantioselectivity improved; in many cases
the ee values were higher than those reported in Table
HP-5MS column (5% phenyl methyl siloxane;
30m · 0.321mm · 0.25lm) on an Agilent 6850 Series
GC System. GC–MS analyses were performed on a
Hewlett–Packard 6890-5793MSD, and microanalysis
on an Elemental Analyzer 1106–Carlo Erba-instrument.
Optical rotations were determined on a Perkin–Elmer
model 341 polarimeter (c = 1g/100mL, CHCl3).
1
. A significant increase in the enantioselectivity was
again obtained with K. marxianus, by which it was pos-
sible the preparation of almost one out of the four
stereoisomers: ethyl (2R,3S)-2-(4-chlorophenoxy)-3-
hydroxybutanoate. The use of S. cerevisiae CBS 7336
caused a remarkable increase of eesyn = 63% when com-
pared to 2% obtained using growing cells. When using
the resting cells of T. variabilis, (2R,3S)-2 was almost
the only stereoisomer formed within the syn pair
The eeÕs and absolute configurations of the reaction
products were determined by HPLC analysis performed
on a Perkin–Elmer 200 series with a UV/vis detector
785A on a commercially available Chiralcel OD (Daicel)
in isocratic conditions employing n-hexane–2-propa-
nol = 98:2, flow rate 0.8mL/min, k = 230nm.
(
(
ee = 98%), while a much lower enantiomeric excess
ee = 42%) was obtained using growing cells.
The absolute configuration of (2R,3S)-3 was established
by single crystal X-ray analysis (see below). Absolute
configurations of 2 were assigned by HPLC under the
3
9
3. Conclusion
conditions used to determine the ee values.
Growing and resting cells of several yeasts were screened
to find out the best biosystem to diastereo- and enantio-
selectively reduce the ethyl 2-(4-chlorophenoxy)-3-oxo-
4.2. Chemicals
Compound 1 was prepared by reacting ethyl 2-chloro-3-
oxobutanoate (Aldrich Chemical Co.) and caesium 4-
chlorophenate. Caesium 4-chlorophenate was prepared
by adding to a solution of 4-chlorophenol (10.51g,
8.17mmol) in absolute ethanol (15mL) a suspension of
Cs CO (8.17mmol) in absolute EtOH (35mL). The
butanoate
1
hydroxybutanoate 2.
into ethyl 2-(4-chlorophenoxy)-3-
Kluyveromyces marxianus was found to be the best
microorganism for accomplishing such a transforma-
tion, as it nearly afforded enantiomerically pure
2
3
mixture was stirred for 3h at room temperature. EtOH
was then removed under reduced pressure and a white
powder was obtained.
(
2R,3S)-2. Its absolute configuration was unambigu-
ously established by single crystal X-ray analysis of the
corresponding acid 3, quantitatively obtained by hydro-
lysis of (2R,3S)-2 isolated from the reduction reaction
performed in the presence of K. marxianus.
4.2.1. Preparation of ethyl 2-(4-chlorophenoxy)-3-oxo-
butanoate 1. Ethyl 2-chloro-3-oxobutanoate (18.22
mmol) and caesium 4-chlorophenate (25.52mmol), were
stirred at 50ꢁC. The reaction was stopped after 4h (GC
analysis). The reaction mixture was treated with ethyl
acetate and then washed with saturated aq NaCl. The
organic extract was dried over anhydrous Na SO and
the solvent evaporated under reduced pressure. The
product was isolated by chromatography (silica gel;
mobile phase: petroleum ether/ethyl acetate = 9:1);
Work is currently in progress aimed at producing the
four enantiomerically pure stereoisomers of 2 by screen-
ing a higher number of known and not yet reported
microorganisms with reducing activity. Furthermore, at-
tempts to isolate the enzyme responsible of K. marxianus
reductase activity and having a reverse stereochemical
preference with respect to the already reported isolated
2
4
4
4
1
enzyme are also underway.
69% yield; keto form/enol form = 68:32 by H NMR .
Oil. IR(neat): 3600–3150, 3100, 3073, 2985, 2940,
2
1371, 1268, 1216, 1167, 1089, 1013, 827, 665cm . H
875, 1758, 1735, 1661, 1629, 1587, 1489, 1446, 1414,
À1
1
4. Experimental
NMR(CDCl ): d 11.32 (s, 1H, OH enol: exchanges with
D O); 7.26–7.20 (m, 4H, aromatic protons, 2H of keto
2
3
4
.1. General
form and 2H of enol form), 6.87–6.81 (m, 4H, aromatic
protons, 2H of keto form and 2H of enol form), 5.03 (s,
1H, CH), 4.30–4.23 (q, J = 7.14Hz, 2H, CH CH , keto
Melting points were taken on electrothermal apparatus
and are uncorrected. H NMRspectra were recorded
1
2
3
in CDCl on a Varian Mercury 300MHz spectrometer
3
form), 4.22–4.15 (q, J = 7.14Hz, 2H, CH CH , enol
2 3
with chemical shifts reported in parts per million (d).
Absolute values of the coupling constant (J) are
reported. IRspectra were also recorded on a Perkin–
Elmer 681 spectrometer. Reaction progress was moni-
tored by TLC or GC analyses. Thin-layer chromatogra-
phy (TLC) was performed on silica gel sheets with a
fluorescent indicator (Statocrom SIF, 60 F254 Merk);
TLC spots were observed under ultraviolet light or
visualized with I2 vapour. Column chromatography
was conducted using silica gel Merk 60 (0.063–
form), 2.35 (s, 3H, COCH , keto form); 1.95 (s, 3H,
3
COCH , enol form), 1.29–1.24 (t, J = 7.14Hz, 3H,
3
CH CH , keto form); 1.17–1.13 (t, J = 7.14Hz, 3H,
3
2
1
3
CH CH , enol form). C NMR(CDCl 3): d 200.66,
3
2
165.88, 155.44, 129.95, 129.63, 127.87, 116.71, 116.05,
83.16, 62.79, 26.56, 14.22. GC–MS (70eV) (m/z) (rel.
3
7
+
35
+
int.) 258 [M( Cl) , 10], 256 [M( Cl) , 29], 216 (10),
214 (30), 147 (15), 143 (32), 141 (100), 139 (29), 129
(8), 128 (9), 113 (11), 111 (22), 75 (15), 43 (37). Anal.
Calcd for C H ClO : C, 56.25; H, 5.08. Found: C,
1
2
13
4
0
.200lm). GC analyses were performed by using a
56.21; H, 5.06.

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3511–3517
3515
4
.3. Microorganism sources
monitored by GC analysis and stopped at times indi-
cated in Table 2. The contents of the flask were then cen-
trifuged and the supernatant extracted with ethyl
acetate. All the reactions were repeated at least twice
without any noticeable change in the results.
K. marxianus CBS 6556, S. cerevisiae DSM 11285,
S. cerevisiae CBS 7336, Cr. curvatus ATCC 29509,
C. bombicola ATCC 22214 and T. variabilis DSM
7
(
0714 were obtained from public type culture collections
ATCC, CBS, DSM).
4.5.4. Ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate
2
.
Oil. IR(neat): 3500–3100, 3061, 2982, 2933, 1738,
1596, 1584, 1492, 1375, 1283, 1238, 1200, 1094, 1023,
4
.4. Culture medium
À1
1
825cm . H NMR(CDCl ): d 7.26–7.18 (m, 4H, aro-
3
The microorganisms were cultivated under aerobic
conditions in a medium containing 0.3% yeast extract,
matic protons, 2H for each stereoisomer pair), 6.88–
6.78 (m, 4H, aromatic protons, 2H for each stereoisomer
pair), 4.56–4.53 (d, J = 4.29Hz, 1H, CHOC H Cl of the
0
.3% malt extract, 0.5% peptone and 1% glucose. To
6
4
the same medium, 2% Agar-Agar was added for cells
preservation on agar slants.
anti pair), 4.43–4.41 (d, J = 4.94Hz, 1H, CHOC H Cl of
6 4
the syn pair) 4.30–4.17 (m, 6H, 3H for each pair of
stereoisomers: 1H of CHCH3 and 2H of CH CH ),
2.90–2.50 (br s, 2H, 1H for each pair of stereoisomers,
2
3
4
4
.5. Bioreduction methodologies
OH: exchange with D O), 1.34–1.32 (d, J = 6.45Hz,
2
.5.1. BakerÕs yeast-induced bioreduction. BakerÕs yeast
3H, CHCH3 of the anti pair), 1.33–1.31 (d,
J = 6.45Hz, 3H, CHCH of the syn pair), 1.25–1.21 (t,
(
2.5g) was dispersed to give a smooth paste in tap
3
water (4.5mL). Ethyl 2-(4-chlorophenoxy)-3-oxobut-
anoate 1 (25mg) dissolved in ethanol (0.5mL) was
added and stirred at 30ꢁC in a LAB-LINE orbital
shaker (250rpm). The initial pH was 4.16. The reaction
progress was monitored by GC. After three hours, the
reaction was stopped by centrifugation, decantation
and extraction by EtOAc. The extracts were dried over
anhydrous Na SO and the solvent evaporated under
J = 7.14Hz, 3H, CH CH of the anti pair), 1.24–1.20
3
2
1
3
(t, J = 7.14Hz, 3H, CH CH of the syn pair).
3
C
2
NMR(CDCl 3): d 169.76, 156.45, 129.76, 127.29,
116.88, 81.52, 81.06, 68.65, 68.60, 61.95, 19.10, 14.37.
3
GC–MS (70eV) (m/z) (rel. int.) 260 [M( Cl) , 5], 258
7
+
3
5
+
[M( Cl) , 17], 214 (21), 143 (30), 141 (100), 139 (16),
130 (17), 128 (52), 111 (13), 75 (12), 43 (13). Anal. Calcd
for C H ClO : C, 55.81; H, 5.81. Found: C, 55.84; H,
2
4
12 15
4
reduced pressure. A yellow oil was obtained.
5.83.
4
.5.2. Bioreduction using growing cells. Cells preserved
4.6. Preparation of (2R,3S)-2-(4-chlorophenoxy)-3-hydr-
oxybutanoic acid 3
on agar slants at 4ꢁC were used to inoculate a 250mL
flask containing the cultivation medium (100mL). The
flask was incubated aerobically at 30ꢁC on an orbital
shaker (250rpm). The 250mL flask containing the culti-
vation medium (100mL) was then inoculated with the
To (2R,3S)-2 (1.357mmol) in THF (18mL), a solution
of KOH (152mg, 2.713mmol, in 6mL water) was added.
The resulting mixture was stirred at room temperature
for 1h. Reaction progress was monitored by GC analy-
sis. THF was then removed under reduced pressure and
the alkaline solution washed three times with ethyl ether,
acidified with 2M HCl and extracted three times with
ethyl ether. The second extract was combined and dried
over anhydrous Na SO . The solvent was evaporated
2
4h old suspension (5mL) and incubated under the
same conditions for 24h. A 1L flask containing the same
cultivation medium (400mL) was then inoculated with
the latter suspension (5mL) and incubated for 24h.
Ethyl 2-(4-chlorophenoxy)-3-oxobutanoate 1 (200mg)
dissolved in ethanol (1mL) was then added to the cul-
ture. Reaction progress was monitored by GC analysis
and stopped at the times indicated in Table 1. The con-
tent of the flask was then centrifuged and the superna-
tant extracted with ethyl acetate. All the reactions
were repeated at least twice without any noticeable bias
in the results.
2
4
under reduced pressure and a colourless oil obtained.
The crude product was purified by crystallization (chlo-
roform/hexane). Quantitative yield. Mp: 105–106ꢁC
20
(CHCl /hexane). ½aŠ ¼ þ12:4 (c 1.0, CHCl ). IR
3
D
3
(KBr): 3700–3200, 3014, 2981, 2926, 2855, 1706, 1593,
1584, 1493, 1374, 1286, 1241, 1175, 1132, 1110, 1089,
À1
075, 1018, 886, 829cm . H NMR(500MHz, CDCl ):
3
1
1
4
.5.3. Bioreduction using resting cells. Culture condi-
d 7.22–7.21 (m, 2H, aromatic protons), 7.20–7.00 (br s,
tions were the same as in the case of the growing cells.
Once cell growth reached the stationary phase, cells
were harvested by centrifugation (20min and
2H, COOH and OH: exchange with D O), 6.82–6.80
2
(m, 2H, aromatic protons), 4.48–4.47 (d, J = 3.58Hz,
1H, CHOC H Cl), 4.36–4.29 (qd, J = 6.33Hz and
3.58Hz, 1H, CHOH), 1.37–1.36 (d, J = 6.33Hz, 3H,
6
4
4
000rpm) at 4ꢁC. The cells were then recovered and
1
3
washed three times (20mL each time) with 0.1M
KH PO buffer pH = 7. The cells were sedimented by
CH CHOH). C NMR(76MHz, CDCl 3): d 173.38,
3
156.04, 129.61, 127.33, 116.57, 80.33, 68.54, 19.09. Anal.
Calcd for C H ClO : C, 52.07; H, 4.81. Found: C,
52.06; H, 4.85.
2
4
centrifugation as previously described. The cell wet
mass, free from culture medium, was re-suspended in
the same buffer solution enriched with 1% glucose to
reach a concentration of 0.5g/L. Then, substrate 1
1
0
11
4
4.7. X-ray analysis
(
medium. The flask was shaken (250rpm) in an orbital
shaker and kept at 30ꢁC. The reaction progress was
2mM final concentration) was added to the reaction
To establish the absolute configuration at C(7) and C(9)
in an unambiguous manner, suitable crystals were

3516
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3511–3517
grown and subjected to single crystal X-ray analysis,
using a Nonius Kappa CCD area detector diffracto-
meter equipped with a fine focus sealed graphite-mono-
chromated Mo-Ka radiation (k = 0.71073A). Data
for (2R,3S)-2-(4-chlorophenoxy)-3-hydroxybutanoic
acid 3 were collected at 293(2)K. Data reduction and
Roger, P. Tetrahedron: Asymmetry 2001, 12, 2185–
189.
. Yong, K. H.; Chong, J. M. Org. Lett. 2002, 4, 4139–
4142.
. Chan, K. F.; Wong, H. N. Org. Lett. 2001, 3, 3991–3994,
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0. Khripach, V. A.; Zhabinskii, V. N.; Fando, G. P.; Kuchto,
A. I.; Lyakhov, A. S.; Govorova, A. A.; Groen, M. B.; Van
Der Louw, J.; De Groot, A. Steroids 2004, 69, 495–
499.
2
8
9
˚
1
cell refinement were carried out with the programs
4
5
46
DENZO and COLLECT. The structure was solved
4
7
by the direct methods procedure of SIR97, while the
refinement processes were carried on full matrix least
squares technique using SHELXL-97. Detailed crystal
data and geometrical parameters have been deposited in
the supporting information (cif file). The asymmetric
unit of (2R,3S)-2-(4-chlorophenoxy)-3-hydroxybutanoic
acid 3 with the atomic numbering scheme is depicted in
Figure 2.
11. Chen, W.-Y.; Lu, J.; Shen, Z.-X.; Lang, J.-P.; Zhang,
L.-F.; Zhang, Y.-W. Chin. J. Chem. 2003, 21, 192–195,
Chem. Abstr. CAN 138:305760.
4
8
4
9
12. Hu, J.-B.; Zhao, G.; Ding, Z.-D. Angew. Chem., Int. Ed.
2
001, 40, 1109–1111.
1
3. Vinogradov, M. G.; Gorshkova, L. S.; Pavlov, V. A.;
Mikhalev, O. V.; ChelÕtsova, G. V.; Razmanov, I. V.;
Ferapontov, V. A.; Malyshev, O. R.; Heise, G. L. Russ.
Chem. Bull. 2000, 49, 460–465, Chem. Abstr. CAN
Pertinent crystallographic data for (2R,3S)-2-(4-chloro-
phenoxy)-3-hydroxybutanoic acid 3: C H ClO , M =
1
33:252092.
1
0
11
4
r
14. Hu, X.-M.; Liu, J. W. Univ. J. Nat. Sci. 1999, 4, 205–210,
Chem. Abstr. CAN 132:107497.
15. Knowles, W. S.; Sabacky, M. J. J. Chem. Soc., Chem.
Commun. 1968, 1445–1446.
6. Knowles, W. S.; Sabacky, M. J.; Vineyard, B. D. J. Chem.
Soc., Chem. Commun. 1972, 10–11.
7. Kagan, H. B.; Dang, J. P. J. Am. Chem. Soc. 1972, 94,
À3
30.64gcm , orthorhombic, space group: P2 2 2, a =
1 1
2
1
˚
3.2109(4), b = 24.8989(9), c = 6.8380(2)A, cell vol-
˚
3
ume = 2244.75(12)A , Z = 8, T = 293(2)K, qc =
À3
À1
1
1
1
1
.365gcm , l = 0.331mm , h range = 2.25ꢁ–27.52ꢁ,
hkl indices À17 6 h 6 17, À31 6 k 6 32, À8 6 l 6 8,
reflections (measured) = 12,009, reflections (un-
ique) = 5031, reflections (unique [F > 2r{jF j}]): 2563,
6
429–6433.
o
o
8. Carneiro, J. W. M.; Oliveira, C. S. B.; Passos, F. B.;
Aranda, D. A. G.; Souza, P. R. N.; Antunes, O. A. C.
J. Mol. Cat., A: Chem. 2001, 170, 235–243.
R
= 0.052, 344 parameters, R /wR (all data): 0.1433/
1 2
int
0
.1059, R /wR (I > 2r(I)): 0.0529/0.0824, Flack para-
1 2
meter = À0.02(8), largest diff. peak/hole: 0.162/
19. Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr.
Opin. Chem. Biol. 2004, 120–126.
À3
˚
À0.245eA
.
2
0. Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T.
Tetrahedron: Asymmetry 2003, 14, 2659–2681.
2
1. Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.;
Watson, J. D. Molecular Biology of the Cell, 3rd ed.;
Garland: New York and London, 1994.
Acknowledgements
Work carried out under the framework of the National
Project ÔProgettazione, Sintesi e Valutazione Biologica
di Nuovi Farmaci CardiovascolariÕ was supported by
the Ministero dellÕIstruzione, dellÕUniversit a` e della
Ricerca (MIUR, Rome). Thanks are also due to the
University of Bari and CNR-Istituto di Chimica dei
Composti OrganoMetallici-ICCOM/Sezione di Bari
22. McCoy, M. Chem. Eng. News 1999, 77, 10–13.
2
Sinisterra, J. V. Tetrahedron: Asymmetry 2004, 15, 951–
´
3. Carballeira, J. D.; Alvarez, E.; Campillo, M.; Pardo, L.;
9
62.
4. Berger, J.; Moller, D. E. Annu. Rev. Med. 2002, 53, 409–
35.
5. Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.;
Leitersdorf, E.; Fruchart, J. C. Circulation 1998, 98, 2088–
2
2
4
(
Italy).
2
093.
2
2
6. Tikkanen, M. Curr. Opin. Lipidol. 1992, 3, 29–33.
7. Zimetbaum, P.; Frishman, W.; Kahn, S. J. Clin. Pharma-
col. 1991, 31, 25–37.
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