Baker's yeast-mediated reduction of ethyl 2-(4-chlorophenoxy)-3- oxoalkanoates intermediates for potential PPARα ligands

Article abstract of DOI:10.1016/j.tetasy.2004.08.027

Several 2-(4-chlorophenoxy)-3-oxoesters were prepared in fair to good yields and then reduced in the presence of baker's yeast to the corresponding alcohols having de's up to 92% and ee's >99%. The absolute configuration of nearly enantiomerically pure ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate was assigned by both comparison of the sign of the specific rotation and HPLC retention times of authentic samples prepared from threonines. Reduction of ethyl 2-(4-chlorophenoxy)-3-oxo-4-phenylbutanoate afforded only enantiomerically pure ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-4-phenylbutanoate (out of the four possible stereoisomers), whose absolute configuration was established by single crystal X-ray analysis. Furthermore, reduction of ethyl 2-methyl-2-(4-chlorophenoxy)-3-hydroxybutanoate with a quaternary stereogenic carbon (C₂) gave both of the two expected diastereoisomers with ee = 95% and 96%. Insight into the mechanism of baker's yeast-mediated reduction of prochiral ketoesters is also reported.

Full text of DOI:10.1016/j.tetasy.2004.08.027

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3501–3510
BakerÕs yeast-mediated reduction of ethyl
2-(4-chlorophenoxy)-3-oxoalkanoates intermediates for
potential PPARa ligands
Maria Grazia Perrone,a Ernesto Santandrea,a Antonio Scilimati,a,* Vincenzo Tortorella,a
Francesco Capitellib and Valerio Bertolasic
a
`
Dipartimento Farmaco-Chimico, Universita di Bari, Via E.Orabona 4, 70125 Bari, Italy
bIstituto di Cristallografia (IC-CNR), Via Amendola 122/o, 70125 Bari, Italy
c
`
Dipartimento di Chimica and Centro di Strutturistica Diffrattometrica, Universita di Ferrara,
Via Borsari 46, 44100 Ferrara, Italy
Received 26July 2004; accepted 20 August 2004
Abstract—Several 2-(4-chlorophenoxy)-3-oxoesters were prepared in fair to good yields and then reduced in the presence of bakerÕs
yeast to the corresponding alcohols having deÕs up to 92% and eeÕs >99%. The absolute configuration of nearly enantiomerically
pure ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate was assigned by both comparison of the sign of the specific rotation and HPLC
retention times of authentic samples prepared from threonines. Reduction of ethyl 2-(4-chlorophenoxy)-3-oxo-4-phenylbutanoate
afforded only enantiomerically pure ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-4-phenylbutanoate (out of the four possible stereoiso-
mers), whose absolute configuration was established by single crystal X-ray analysis. Furthermore, reduction of ethyl 2-methyl-
2-(4-chlorophenoxy)-3-hydroxybutanoate with a quaternary stereogenic carbon (C2) gave both of the two expected diastereoisomers
with ee = 95% and 96%. Insight into the mechanism of bakerÕs yeast-mediated reduction of prochiral ketoesters is also reported.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
BakerÕs yeast is often chosen, with respect to other
microorganisms with reducing activity, as it is easy to
use and does not require the presence of expensive
cofactors to exert its action, is not pathogenic, is versa-
tile and has no environmental impact.
The asymmetric reduction of prochiral ketones repre-
sents one of the most used methods for preparing chiral
alcohols in very high enantiomeric excess.1,2 This trans-
formation can be accomplished by using various chiral
reducing agents3,4 or chirally modified boron- and alu-
minium-hydrides,5,6 through enantioselective reduction
by hydride transfer from carbon or nitrogen and by
catalytic reduction with chiral transition metal com-
plexes.7–12
Herein bakerÕs yeast has been used to prepare optically
active clofibrate analogues. Fibrates constitute a widely
used class of lipid-modifying agents. Treatment with
fibrates results in a substantial decrease in plasma tri-
glycerides and is usually associated with a moderate
decrease in LDL and an increase in HDL cholesterol
concentration.21
Biotransformations have also extensively been used for
the preparation of chiral alcohols.13 In particular, li-
pase-mediated kinetic resolution of racemic alcohols,14
asymmetric reduction of the carbonyl of a prochiral ke-
tone by bakerÕs yeast15–18 and hydroxylation reaction19
have widely been used to prepare enantiomerically pure
alcohols.20
Over the course of our previous investigation, optically
active, acyclic and cyclic (rigid) analogues of clofibrate,
were prepared by asymmetric synthesis,22 crystallization
of their diastereomeric salts with chiral amines23 and by
kinetic resolution performed in the presence of lip-
ases.15,24 We have also investigated for a long time the
pharmacological activity of clofibric acid derivatives in
order to find and, possibly, dissociate the structural
determinants of the different effects.25–28 Hence, the first
*
Corresponding author. Tel.: +39 080 5442762; fax: +39 080
5442231; e-mail: ascilimati@farmchin.uniba.it
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.08.027

3502
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
CO2Et
O
Cl
Clofibrate
Cl
CO2Et
R1
O
CO2Et
R2
O
CO2Et
CO2Et
R1
R2
R1
O
Cl
HO
R1
Cl
O
O
2
1
Figure 1. Clofibrate molecular structure and retrosynthesis of ethyl 2-(4-chlorophenoxy)-3-hydroxyalkanoates 2a–g.
part of our study consisted of the synthesis of more
functionalized optically active analogues of ethyl aryl-
oxyalkanoates capable of eventually giving additional
interactions with the target receptor ligand-binding
domain.29
2. Results and discussion
A number of racemic clofibrate analogues were synthe-
sized29 (Fig. 1) by reacting the chloroketoesters, pre-
pared from commercially available ketones, with
caesium 4-chlorophenate at 50ꢁC to give the 4-chloro-
phenoxyketoesters 1a–g, which in turn were reduced to
the corresponding desired alcohols 2a–g by NaBH4/
methanol at 0ꢁC. Compound 1g was prepared by treat-
ing 1a with CH3I/Cs2CO3. To prepare the same com-
pounds 2a–g in optically active form, the last step of
Figure 1 was performed in the presence of bakerÕs
yeast/water at 30ꢁC. The results of the reduction of
the prochiral ketoesters 1a–g to the corresponding
hydroxyesters 2a–g by bakerÕs yeast (Scheme 1) are sum-
marized in Table 1.
Such clofibrate analogues have been synthesized in their
racemic form (Fig. 1) and pharmacologically evaluated,
by a transactivation assay, as potential peroxisome pro-
liferator-activated receptor isoform a (PPARa) agonists.
Unfortunately, they were unable to interact with the
PPARa ligand binding domain. Among the several rea-
sons for a lack of activity, one could be that they were
tested as a mixture of the four possible stereoisomers,
these compounds having two stereogenic centres.
The last step of the previously used synthetic pathway
consisted of the reduction of the carbonyl group with
NaBH4 (Fig. 1). The diastereomeric excesses obtained
ranged between 2% and 60% (Table 1).
Reduction rates depended upon the substrate concentra-
tion and the size of R1. Substrate concentration (5.0g/L
for 1a, 1.7g/L for 1f, 1.0g/L for 1b–d and 1g) was chosen
to have reasonable conversion rates. In the case of 1e, a
very small amount of product was formed (about 2%
within 9days) even at a substrate concentration of
0.05g/L. Compound 1a (R1 = H) was the fastest reacting
compound; within 3h it was completely converted into
the product 2a with very high diastereoselectivity
(de = 92%, vs 11% obtained in the reduction conducted
with NaBH4) with the major stereoisomer formed being
Herein, the results of the bakerÕs yeast-mediated asym-
metric reduction of 2-(4-chlorophenoxy)-3-oxoalkano-
ates 1a–g to 2a–g are reported. Such a method was
selected in an attempt to prepare optically active or even
better enantiomerically pure 2-(4-chlorophenoxy)-3-
hydroxyalkanoates 2a–g suitable for performing the
bioassay.
Table 1. Results of the reduction of 1a–g performed in the presence of bakerÕs yeast
Substr. concn (g/L) Reaction time (day) Dea (%) syn > anti Eesynb(%) Eeanti (%) Conversionc (%)
b
Substrate R1
R2
1a
1b
1c
1d
1e
1f
H
H
H
H
H
H
CH3
C2H5
5.0
1.0
1.0
1.0
0.05
1.7
1.0
3h
5
92 (11)
50 (7)
94
51
7
Quantitative
88
3
n-C3H7
i-C3H7
t-C4H9
C6H5
1.5
4
89 (22)
70 (25)
Ndd (8)
>99 (34)
2 (65)
89
5
Quantitative
43
70
33
Ndd
—
95
9
Ndd
>99
>99
2
3
Quantitative
Quantitative
1g
CH3 CH3
2
a Diastereomeric excesses of products 2a–g, determined by 1H NMR. In brackets are de values obtained reducing 1a–g with NaBH4.
b Enantiomeric excesses of 2a–g determined by HPLC using a Daicel OD column packed with chiral stationary phase.
c Conversion = % substrate 1a–g transformed into product 2a–g, determined by 1H NMR and GC analysis.
d Nd = not determined, due to low conversion (about 2%).

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
3503
O
O
CO2Et
R2
O
CO2Et
R2
Baker's yeast
Cl
R1
Cl
HO
R1
1
2
R1 = H; R2 = CH3, C2H5, n-C3H7, i-C3H7, t-C4H9, C6H5
R1 = R2 = CH3
Scheme 1. BakerÕs yeast reduction of 1a–g to afford 2a–g.
Figure 2. ORTEP view of the asymmetric unit with the atomic numbering scheme of ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-3-
phenylpropanoate 2f. Thermal ellipsoids probability level at 30%.
the syn-(2R,3S) with ee = 94%.30 The reaction times to
reduce 1b–g (43%–100% extent of conversion) were
higher than 24h (1.5–5days). Compound 1c was
completely converted in 1.5days with good diastereo-
and enantioselectivity (de = 89% and eesyn = 89%,
respectively).
The absolute configuration of (2R,3S)-2f was estab-
lished by a single crystal X-ray analysis (Fig. 2).32–36
3. Investigations into the mechanism of bakerÕs yeast-
mediated reduction of prochiral ketones 1a–g
Lower conversion (88% and 43%, respectively) and ster-
eoselectivity (de = 50% and 70%, eesyn = 51% and 70%,
respectively) were obtained incubating 1b and 1d in the
presence of bakerÕs yeast.
All compounds 1a–f are present in the reaction medium
in both enol- and keto-forms as can be seen from their
1H NMR spectra. The extent of enolization varies
slightly from one compound to the other (40–60%).
Compound 1e (R1 = t-Bu) gave, as above mentioned,
only 2% of alcohol 2e. This very low extent of conver-
sion does not seem to only be due to the steric hindrance
exerted from the t-butyl group, but it also appears to be
a consequence of different factors if the results obtained
reducing 1e and 1f (R1 = t-Bu and phenyl, respectively)
are compared. In fact, a phenyl ring (1f) and a t-butyl
(1e) have a similar Van der Waals volume: 45.84cm3/
mol and 44.34cm3/mol, respectively.31 Conversely for
1e, which is almost unreactive under the conditions
used, 1f was completely reduced within 3days and with
the highest observed diastereo- and enantioselection
[de >99% and eesyn >99%, the (2R,3S) stereoisomer
being only formed]. These results definitively suggest
that bakerÕs yeast-mediated bioreduction of compounds
such as 1a–g, are strongly affected by both electronic
features and group size bonded to the prochiral
carbonyl.
Reduction of racemic a-substituted b-ketoesters poses a
problem for diastereo- and enantioselection (the case of
1f is depicted in Figure 3 as an example). Under the con-
ditions of the yeast reduction, the b-ketoester group can
enolize (see above), which for 2-substituted compounds,
such as 1a–f, results in racemization. Hence, racemic 2-
substituted 3-oxoalkanoates 1a–f can be converted
under particular circumstances (see below) into a
single enantiomer of the corresponding 2-substituted 3-
hydroxyalkanoates 2a–f. Reduction can in principle
occur by enantiotopic face differentiating in the hydro-
genation of the carbon–carbon double bond of the enol
intermediate; in this case, in analogy to the reduction of
an activated carbon–carbon double bond, anti-products
should be expected.37
On the other hand, activated tetrasubstituted carbon–
carbon double bonds are not usually reduced by bakerÕs

3504
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
O
O
COOEt
O
COOEt
O
COOEt
Cl
Cl
HO
Cl
HO
1f
anti-(2R,3R)-2f
syn-(2S,3R)-2f
O
COOEt
O
COOEt
O
COOEt
Cl
O
Cl
HO
Cl
O
(2S)-1f
(2R)-1f
O
COOEt
O
COOEt
Cl
HO
Cl
HO
anti-(2S,3S)-2f
syn-(2R,3S)-2f
Figure 3. Possible reduction products of 1f and its keto–enol equilibrium.
yeast, otherwise fast reduction of one of the two
enantiomeric forms of the substrates, in its keto form,
will drive the reaction towards the selective formation
of only one out of the four possible stereoisomers,
provided that difference in rate between racemization
of the wrong enantiomer and reduction of the right
enantiomer is large enough.17
proposed mechanism, because ethyl 2,2-dimethylaceto-
acetate might simply not be reduced probably for its
non-acceptance in the enzyme catalytic site due to other
factors.
The first evidence that the reduction might not undergo
through the hydrogenation of the carbon–carbon dou-
ble bond of the enol form comes from the reduction of
compounds 1a–f in which the main stereoisomer is
the syn-form. This allowed us to hypothesize that
the reaction proceeds through the reduction of the
carbonyl.17
In particular, in our case the syn-(2R,3S) is the major
(2a) or the unique (2f) stereoisomer formed at least in
the reduction of 1a and 1f for which the absolute config-
urations of the corresponding product, 2a and 2f, were
established (Table 1).
Additionally, the reaction of the non-enolizable 3-oxo-
ester 1g provides further evidence as the reduction took
place and this supports the mechanism detailed in
Figure 3.
Deol et al. while exploring this reaction,37 in a study
aimed at differentiating between these two possibilities,
incubated the non-enolizable b-keto ester ethyl 2,2-
dimethylacetoacetate in the presence of bakerÕs yeast.
However, it was not reduced (Scheme 2). This could sug-
gest that the reduction of non-enolizable 3-oxoesters,
such as ethyl 2,2-dimethylacetoacetate, does not occur,
because the reaction should take place on the enol form.
Unfortunately, this evidence is not proof enough for the
As expected, no diastereomeric excess (2%) was re-
corded because no interconversion between the enantio-
meric forms is possible unlike for 1a–f. In contrast,
enantioselectivity is very high (ee >99% and 95%): only
one enantiomer within each couple of possible stereo-
isomers is formed. Furthermore, the absence of dia-
stereoselection observed, confirms that bakerÕs yeast
stereoselectivity is completely unaffected by the stereo-
chemistry of the groups bound to the carbonyl.38 In
the reaction mixture, compound 1g is racemic and each
enantiomer is completely reduced to form the two
almost enantiomerically pure stereoisomers 2g (Scheme
3). This means that the newly formed stereogenic centre
has the same absolute configuration both starting from
CO2Et
CO2Et
Baker's yeast
*
O
HO
Scheme 2. Absence of reaction of ethyl 2,2-dimethylacetoacetate in the
presence of bakerÕs yeast.

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
3505
O
CO2Et
O
CO2Et
*
Baker's yeast
*
Cl
O
Cl
HO
(R,S)-1g
2g
quantitative yield; de = 5%
eemajor pair > 99%, eeminor pair = 95%
Scheme 3. Reduction of ethyl 2-methyl-2-(4-chlorophenoxy)-3-oxobutanoate 1g in the presence of bakerÕs yeast.
O
CO2Et
Cl
HO
O
CO2Et
O
CO2Et
Baker's yeast
Cl
AcO
Cl
HO
O
O
CO2Et
2a
3
Cl
Scheme 4. Reaction of 3 in the presence of bakerÕs yeast.
(R)-1g and (S)-1g, otherwise we would have observed
lower eeÕs and a higher de. Hence, considering the pre-
ferred direction of the hydride attack (in the presence
of bakerÕs yeast and on the base of the stereochemical
outcome at C3 of 2a and 2f, if it is the same reductase
enzyme involved) for b-ketoesters, we should have pre-
pared (2R,3S)- and (2S,3S)-2g.39
and the unmodified substrate was recovered from the
reaction medium (Scheme 5).
4. Conclusion
In summary, we have explored the reductive capabilities
of bakerÕs yeast towards compound 1a–g. The results
obtained show that this is a useful tool for preparing a
set of optically active clofibrate analogues in good yields
and with high enantiomeric excesses. In particular, the
best results were obtained with compound 1g, which
was reduced with the highest diastereo- and enantiose-
lectivity, and 1f, whose reduction afforded only one
enantiomerically pure isomer out of the four possible
stereoisomers.
To support definitively the hypothesis that the reduction
occurs on the carbonyl form, we have prepared 3 (the
enol acetate of 1a). In the presence of bakerÕs yeast, it
gave the same results obtained incubating directly 1a.
This means that it was presumably first hydrolysed, by
hydrolases contained in the yeast, and then reduced by
the reductase enzyme to 2a (Scheme 4). Once again, this
finding represents further confirmation of the above
proposed mechanism of the reduction reaction as the
main products have a syn-conformation, namely that
they could be not formed by direct reduction of the
carbon–carbon double bond of the enol acetate.
Efforts have also been made to clarify the bakerÕs yeast-
mediated reduction reaction mechanism, definitively
demonstrating that bakerÕs yeast-assisted bioreduction
of such 3-oxoesters does not occur on the enol interme-
diate and, that the intermediate itself is not an essential
condition for the reaction to occur.
Next, 4 (methyl enol ether of 1a) was prepared, which in
turn did not undergo hydrolysis during the incubation
with bakerÕs yeast under the same conditions used as
for 1a. No reaction took place after 6days of incubation
Further investigations are currently in progress, aimed
at finding different yeast strains capable of producing
even better results in terms of yield and stereoselectivity
in the reduction of 1a–g, allowing the preparation of the
four possible stereoisomers of 2a–g as well.
O
CO2Et
Baker's yeast
no reaction
Cl
MeO
5. Experimental
4
1H NMR spectra were recorded in CDCl3 on a VAR-
IAN Mercury 300MHz spectrometer and chemical
Scheme 5. Absence of reaction of 4 in the presence of bakerÕs yeast.

3506
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
Table 2. Results of the reduction of ethyl 2-(4-chlorophenoxy)-3-
oxobutanoate 1a to ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate 2a
by using the different reaction medium composition (procedures A–D)
shifts are reported in parts per million (d). Absolute val-
ues of the coupling constant (J) are reported. The extent
of the enolization of 1a–f was measured by 1H NMR. IR
spectra were recorded on a Perkin–Elmer 681 spectro-
meter. Reaction progress was monitored by TLC or
GC analysis. Thin-layer chromatography (TLC) was
performed on silica gel sheets with a fluorescent indica-
tor (Statocrom SIF, 60 F254MERK); TLC spots were
observed under ultraviolet light or visualized with I2
vapour. Column chromatography was conducted using
silica gel MERCK 60 (0.063–0.200lm). GC analyses
were performed by using a HP-5MS column (5% phenyl
Procedure
Reaction
time
Yield
(%)a
De
(%)b
Eemajor (syn)
(%)c
A
5days
5days
3h
93
99
83
87
92
90
92
90
94
95
95
97
B
C
Dd
3h
a Yields were determined on the product isolated by chromatography.
b Diastereomeric excesses were determined by 1H NMR.
c Enantiomeric excesses were determined by HPLC. A syn-conforma-
tion was established by comparison with HPLC chromatograms of
authentic samples prepared from threonines.30
d Procedure D was used to reduce 1b–f, because it was faster than
procedure A and B. Comparing, instead procedure C and D, it is
evident that the presence of sucrose in the reaction medium did not
produce (in this case) any effect. In fact, yields, diastereomeric
excesses and enantiomeric excesses had almost the same values.
methyl siloxane; 30m · 0.321mm · 0.25lm) on
a
Agilent 6850 SERIES GC SYSTEM. GC–MS analyses
were performed on a HEWLETT PACKARD 6890–
5793MSD, and microanalysis on a Elemental Analyzer
1106-Carlo Erba-instrument. Optical rotations were
determined on a Perkin–Elmer model 341 polarimeter;
determinations were performed in CHCl3, c = 1g/
100mL.
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-propan-
ol = 98:2, flow rate 0.8mL/min, k = 230nm.
Table 3. Medium composition in the reduction reaction of ethyl 2-(4-
chlorophenoxy)-3-oxoalkanoates 1a–g by using procedure D
Compound Substrate
BakerÕs yeast EtOH
Reaction
(%, v/v) time (day)
concentration (g/L)
(g/L)
1a
1b
1c
1d
1e
1f
5
0.48
0.33
0.7
2
3 h
1
65
2
All chemicals and solvents were purchased from Aldrich
Chemical Co.
1
1.5
4
1
0.7
2
0.05
1.7
1
100a
1.21
0.2
2
9
5.1. BakerÕs yeast-mediated reduction of ethyl
2-(4-chlorophenoxy)-3-oxoalkanoates 1a–g
3.5
62
2
1g
a A further 100g of bakerÕs yeast were added to reaction medium after
2.5days. Similarly, after 6days.
5.1.1. Procedure A. BakerÕs yeast (2.5g) was dispersed
to give a smooth paste in tap water. Ethyl 2-(4-chloro-
phenoxy)-3-oxoalkanoate was then added and stirred
at 37ꢁC and 250rpm. The reaction progress was moni-
tored by GC analysis and stopped at the time indicated
in Tables 2–5. The reaction mixture was extracted
several times with EtOAc. The extracts were dried over
anhydrous Na2SO4 and the solvent evaporated under
reduced pressure. A yellow oil was obtained.
Table 4. Medium composition of ethyl 2-methyl-2-(4-chlorophenoxy)-
3-oxobutanoate 1g reduction reaction by using different procedures
Procedurea BakerÕs
yeast (g) (g)
Substrate H2O Sucrose EtOH
(mL) (g)
(mL)
A
B
25
25
75
75
0.2
0.2
0.4
0.3
150
150
47
—
—
—
3
3.3
4.3
—
The reduction reaction was also carried out under
slightly modified versions of procedure A: procedure
B, C and D.
C
D
47
3
a For procedures A, B, C and D, see Section 5.1.
5.1.2. Procedure B. Same as procedure A, but the reac-
tion mixture with sucrose was incubated at 30ꢁC and
250rpm for 30min before adding the substrate.
Table 5. Results of ethyl 2-methyl-2-(4-chlorophenoxy)-3-oxobuano-
ate 1g reduction reaction by using different procedures
5.1.3. Procedure C. Same as procedure B, but the sub-
strate was dissolved in EtOH.
Procedure Reaction Yield De
(%)a
Eemajor pair Eeminor pair
time
(day)
(%)b (%)c (%)c
5.1.4. Procedure D. Same as procedure A, but the sub-
strate was dissolved in EtOH.
A
B
5
4
8
8
85
80
20
21
5
2
3
2
>99
90
96
81
96
C
D
97
9695
5.2. Ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-
butanoate 2a
a Yields were determined on the product isolated by chromatography.
b Diastereomeric excesses were determined by 1H NMR.
c Enantiomeric excesses were determined by HPLC.30
20
From procedure A. Oil. ½aŠ ¼ þ33:1 (c 1.0, CHCl3).
D
De = 92%. Ee = 94%. IR (neat): 3600–3200, 3056,

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
3507
2985, 2932, 2855, 1748, 1596, 1491, 1376, 1266, 1236,
couple); 4.19–4.10 (2q, J = 7.14Hz, 4H, CH2O, 2H for
each stereoisomer couple); 4.09–3.95 (m, 2H, CHOH,
1H for each stereoisomer couple); 2.28–2.10 (br s, 1H
for each couple of stereoisomers, OH: exchange with
D2O); 1.65–1.42 (m, 8H, CH2CH2CHOH, 4H for each
stereoisomer couple); 1.27–1.22 (2t, J = 7.14Hz, 6H,
CH3CH2O, 3H for each stereoisomer couple); 0.94–
0.82 (m, 6H, CH3CH2CH2, 3H for each stereoisomer
couple). 13C NMR (CDCl3, d): 169.98, 156.53, 129.73,
116.89, 116.84, 80.61, 80.22, 72.35, 72.23, 61.93, 35.44,
19.00, 14.33, 14.08. GC–MS (70eV) (m/z) (rel int.) 288
[M(37Cl)+, 4], 286[M( 35Cl)+, 12], 216(11), 214 (32),
168 (8), 143 (37), 142 (13), 141 (100), 139 (11), 130
(14), 128 (38), 113 (10), 111 (11), 75 (8), 71 (1), 43
(11). Anal. Calcd for C14H19ClO4: C, 58.74; H, 6.64.
Found: C, 58.73; H, 6.61.
1
1199, 1137, 1095, 1076, 1025, 1009, 826, 738cmÀ1. H
NMR (300MHz, CDCl3, d): 7.25–7.20 (m, 2H, aromatic
protons); 6.85–6.80 (m, 2H, aromatic protons); 4.44–
4.42 (d, J = 4.94Hz, 1H, CHOC6H4Cl); 4.30–4.22 (qd,
J = 7.15Hz and 1.64Hz, 2H of CH2CH3 completely
overlapped to the signal of CHOH); 2.82–2.44 (br s,
1H, OH: exchanges with D2O); 1.35–1.33 (d,
J = 6.45Hz,
3H,
CH3CHOH);
1.27–1.22
(t,
J = 7.15Hz, 3H, CH3CH2). 13C NMR (76MHz, CDCl3,
d): 169.88 (1C, CO); 156.48 (1C, aromatic carbon);
129.68 (2C, aromatic carbons); 127.14 (1C, aromatic
carbon); 116.80 (2C, aromatic carbons); 81.15 (1C,
CHOC6H4Cl) 68.57 (1C, CHOH); 61.85 (1C, CH2CH3);
18.60 (1C, CH3CHOH); 14.33 (1C, CH3CH2). GC–
MS (70eV) (m/z) (rel int.): 260 [M(37Cl)+, 6], 258
[M(35Cl)+, 19], 214 (19), 168 (9), 167 (8), 143 (32), 142
(8), 141 (100), 139 (15), 130 (16), 129 (10), 128 (49),
111 (10), 99 (7), 75 (10), 45 (7), 43 (9). Anal. Calcd for
C12H15ClO4: C, 55.81; H, 5.81. Found: C, 55.84; H,
5.83.
5.5. Ethyl 2-(4-chlorophenoxy)-3-hydroxy-4-methyl-
pentanoate 2d
Oil. ½aŠ ¼ þ0:5 (c = 1.0, CHCl3). 20% Yield. De = 70%.
D
Ee = 70% (of the major stereoisomer pair), ee = 33% (of
the minor stereoisomer pair). IR (neat): 3500–3100,
2926, 2848, 1739, 1491, 1462, 1375, 1237, 1162, 1130,
5.3. Ethyl 2-(4-chlorophenoxy)-3-hydroxypentanoate 2b
20
Oil. ½aŠ ¼ þ5:2 (c 1, CHCl3). 32% Yield. De = 50%.
1
1097, 953, 874, 800, 860cmÀ1. H NMR (CDCl3, d):
D
Ee = 51% (of the major stereoisomer pair). IR (neat):
3432–3237, 3050, 2964, 2920, 2872, 1730, 1642, 1490,
7.27–7.20 (m, 4H, aromatic protons, 2H for each stereo-
isomer couple); 6.86–6.80 (m, 4H, aromatic protons, 2H
for each stereoisomer couple); 4.67–4.66 (d, J = 3.29Hz,
1H, CHOC6H4Cl of the major stereoisomer couple);
4.63–4.61 (d, J = 5.36Hz, 1H, CHOC6H4Cl of the minor
stereoisomer couple); 4.28–4.21 (2q, J = 7.14Hz, 4H,
CH2CH3, 2H for each stereoisomer couple); 3.86–3.82
(t, J = 5.36Hz, 1H, CHOH of the major stereoisomer
couple); 3.75–3.72 (dd, J = 3.29 and 7.62Hz, 1H,
CHOH of the minor stereoisomer couple); 2.36–2.28
(m, 1H, CH(CH3)2 of one stereoisomer couple); 2.05–
1.97 (m, 1H, CH(CH3)2 of the other stereoisomer
couple); 1.70–1.50 (br s, 1H for each couple of stereoiso-
mers, OH: exchange with D2O); 1.29–1.22 (t,
J = 7.14Hz, 6H, CH3CH2, 3H for each stereoisomer
couple); 1.09–1.03 (2d, J = 6.73Hz, 6H, (CH3)2CH of
the major stereoisomer couple); 1.00–0.92 (2d,
J = 6.73Hz, 6H, (CH3)2CH of the minor stereoisomer
couple). 13C NMR (CDCl3, d): 170.25, 156.00, 129.79,
127.15, 116.67, 78.54, 78.14, 70.78, 61.94, 31.21, 29.38,
27.44, 14.34. GC–MS (70eV) (m/z) (rel int.) 288
[M(37Cl)+, 3], 286[M( 35Cl)+, 9], 216(10), 214 (31), 168
(6), 143 (32), 141 (100), 130 (12), 128 (34), 113 (10),
111 (12), 75 (8), 71 (9), 43 (17), 41 (9). Anal. Calcd for
C14H19ClO4: C, 58.74; H, 6.64. Found: C, 58.74; H,
6.63.
1
1458, 1262, 1216, 1092, 1017, 800, 760cmÀ1. H NMR
(CDCl3, d): 7.18–7.14 (m, 4H, aromatic protons, 2H
for each stereoisomer couple); 6.80–6.71 (m, 4H,
aromatic protons, 2H for each stereoisomer couple);
4.51–4.49 (d, J = 4.54Hz, 1H, CHOC6H4Cl of one
stereoisomer couple); 4.43–4.41 (d, J = 3.98Hz, 1H,
CHOC6H4Cl of the other stereoisomer couple); 4.20–
4.13 (q, J = 7.14Hz, 4H, CH2OCO, 2H for each stereo-
isomer couple); 3.98–3.89 (m, 2H, CHOH, 1H for each
stereoisomer couple); 2.40–2.00 (br s, 1H for each couple
of stereoisomers, OH: exchange with D2O); 1.62–1.57
(m, 4H, CH2CHOH, 2H for each stereoisomer couple);
1.20–1.15 (t, J = 7.14Hz, 6H, CH3CH2O, 3H for
each stereoisomer couple); 0.99–0.92 (m, 6H, CH3CH2-
CHOH, 3H for each stereoisomer couple). 13C NMR
(CDCl3, d): 170.00, 156.60, 129.72, 127.30, 116.84,
80.29, 79.88, 73.92, 73.92, 61.86, 61.78, 26.46, 25.78,
14.32. GC–MS (70eV) (m/z) (rel int.) 274 [M(37Cl)+,
4], 272 [M(35Cl)+, 13], 216(8), 214 (24), 168 (7), 143
(33), 142 (9), 141 (100), 139 (11), 130 (13), 129 (9), 128
(38), 111 (10), 99 (9), 75 (8), 57 (8), 43 (6). Anal. Calcd
for C13H17ClO4: C, 57.35; H, 6.25. Found: C, 57.36; H,
6.27.
5.4. Ethyl 2-(4-chlorophenoxy)-3-hydroxyhexanoate 2c
5.6. Ethyl 2-(4-chlorophenoxy)-3-hydroxy-4,4-dimethyl-
pentanoate 2e
Oil. ½aŠ ¼ þ2:0 (c 1, CHCl3). 53% Yield. De = 89%.
D
Ee = 89% (of the major stereoisomer pair). IR (neat):
3500–3150, 3065, 2961, 2934, 2872, 1737, 1595, 1492,
1465, 1379, 1282, 1238, 1199, 1094, 1075, 1030, 825,
Due to the small amount of 2e formed, it was identified
by comparing the GC–MS spectrum obtained when
reducing 1e by NaBH4 and in the presence of bakerÕs
yeast. Oil. GC–MS (70eV) (m/z) (rel int.) 302
[M(37Cl)+, 3], 300 [M(35Cl)+, 9], 227 (2), 216(13), 214
(39), 168 (6), 143 (35), 141 (100), 139 (8), 130 (15), 128
(33), 111 (11), 57 (27), 41 (10).
1
668cmÀ1. H NMR (CDCl3, d): 7.18–7.13 (m, 4H, aro-
matic protons, 2H for each stereoisomer couple); 6.77–
6.73 (m, 4H, aromatic protons, 2H for each stereoisomer
couple); 4.50–4.49 (d, J = 4.40Hz, 1H, CHOC6H4Cl of
the major stereoisomer couple); 4.42–4.40 (d,
J = 3.98Hz, 1H, CHOC6H4Cl of the minor stereoisomer

3508
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
5.7. Ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-3-
phenylpropanoate 2f
ether/ethyl ether = 7:3) as a mixture of the geometric iso-
mers (E/Z = 70:30). Oil. 51% Yield.
Mp = 82.8–83.9ꢁC (by evaporation of ethyl ether), white
5.9.1. (Z)-3. (Table 6). IR: (neat): 3106, 3069, 2984,
2937, 1770, 1724, 1663, 1593, 1488, 1369, 1304, 1215,
20
solid. 33% Yield. De >99%. Ee >99%. ½aŠ ¼ þ27:2 (c
D
1.0, CHCl3). IR (KBr): 3550–3250, 3225, 2955, 2926,
2845, 1733, 1596, 1582, 1490, 1450, 1375, 1261, 1235,
1180, 1090, 1068, 1012, 829cmÀ1 1H NMR (CDCl3,
.
d): 7.25–7.22 (m, 2H, aromatic protons); 6.97–6.94 (m,
2H, aromatic protons); 4.14–4.06(q, J = 7.14Hz, 2H,
OCH2CH3); 2.25 (s, 3H, CH3CO); 1.99 (s, 3H,
CH3C@C); 1.14–1.09 (t, J = 7.14Hz, 3H, OCH2CH3).
13C NMR (CDCl3, d): 168.73 (1C, CH3CO); 161.31
(1C, COOEt); 152.68 (1C, CAr); 151.61 (1C, Colefin);
133.90 (1C, CAr); 129.82 (1C, CAr); 123.17 (1C, Colefin);
116.61 (1C, CAr); 61.32 (1C, OC2CH3); 21.07 (1C,
CH3CO); 17.13 (1C, CHs3C@C); 14.15 (1C, CH3CH2).
GC–MS (70eV) (m/z) (rel int.) 300 [M(37Cl)+, 1], 298
[M(35Cl)+, 4], 258 (35), 257 (15), 256(100), 212 (7),
211 (7), 210 (21), 148 (10), 147 (85), 142 (28), 139 (78),
128 (7), 111 (l9), 75 (l4), 43 (89).
1
1216, 1192, 1093, 1050, 1026, 824, 760cmÀ1. H NMR
(CDCl3, d): 7.50–7.16(m, 7H, aromatic protons);
6.80–6.73 (m, 2H, aromatic protons); 5.17–5.15 (d,
J = 5.50Hz, 1H, CHOH); 4.69–4.67 (d, J = 5.50Hz,
1H, CHOC6H4Cl); 4.11–4.04 (m, 2H, CH2CH3); 3.60–
3.20 (br s, 1H, OH: exchange with D2O); 1.09–1.05 (t,
J = 7.14Hz, 3H, CH3CH2). 13C NMR (CDCl3, d):
169.47, 139.12, 129.73, 129.66, 128.73, 128.61, 126.93,
126.89, 117.19, 82.34, 81.45, 75.03, 74.38, 61.82, 14.17.
GC–MS (70eV) (m/z) (rel int.) 322 [M(37Cl)+, 0.1], 320
[M(35Cl)+, 0.2], 302 [M(35Cl)+ À 18, 0.3], 247 (2), 216
(22), 214 (68), 143 (34), 141 (100), 128 (20), 113 (16),
111 (25), 107 (19), 106 (27), 105 (36), 91 (14), 79 (16),
77 (49), 75 (15), 51 (16), 50 (10). Anal. Calcd for
C17H17ClO4: C, 63.25; H, 5.31. Found: C, 63.76; H,
5.33.
5.9.2. (E)-3. (Table 6). IR (neat): 3106, 3069, 2984,
2937, 1770, 1724, 1663, 1593, 1488, 1369, 1304, 1215,
1180, 1090, 1068, 1012, 829cmÀ1 1H NMR (CDCl3,
.
d): 7.21–7.18 (m, 2H, aromatic protons); 6.86–6.83 (m,
2H, aromatic protons); 4.17–4.10 (q, J = 7.14Hz, 2H,
OCH2CH3); 2.40 (s, 3H, CH3CO); 2.05 (s, 3H,
CH3C@C); 1.14–1.09 (t, J = 7.14Hz, 3H, OCH2CH3).
13C NMR (CDCl3, d): 167.62 (1C, CH3CO); 163.04
(1C, COOEt); 156.09 (1C, CAr); 151.61 (1C, Colefin);
133.01 (1C, CAr); 129.55 (1C, CAr); 127.58 (1C, Colefin);
116.97 (1C, CAr); 61.55 (1C, OCH2CH3); 20.75 (1C,
CH3CO); 17.92 (1C, CH3C@C); 14.15 (1C, CH3CH2).
GC–MS (70eV) (m/z) (rel int.) 300 [M(37Cl)+, 2], 298
[M(35Cl)+, 7], 258 (38), 257 (16), 256 (100), 212 (9),
211 (12), 210 (24), 148 (9), 147 (80), 141 (26), 139 (69),
128 (9), 111 (18), 75 (l4), 43 (72) (Tables 6and 7) .
5.8. Ethyl 2-methyl-2-(4-chlorophenoxy)-3-hydroxy-
butanoate 2g
(Tables 4 and 5). Oil. [a]D = +1.1 (c = 1.0, CHCl3) from
procedure A. IR (neat): 3500–3100, 3062, 2983, 2936,
1736, 1593, 1490, 1443, 1376, 1239, 1094, 1047, 1012,
850, 823cmÀ1 1H NMR (CDCl3, d): 7.21–7.18 (m,
.
4H, aromatic protons, 2H for each stereoisomer couple);
6.85–6.82 (m, 4H, aromatic protons, 2H for each stereo-
isomer couple); 4.28–4.21 (q, J = 7.14Hz, 4H, CH2O-
CO, 2H for each stereoisomer couple); 4.18–4.12 (q,
J = 6.41Hz, 2H, CHOH, 1H for each stereoisomer cou-
ple); 2.80–2.40 (br s, 1H for each stereoisomer couple,
OH: exchange with D2O); 1.46(s, 3H, CH3Cq of one
stereoisomer couple); 1.40 (s, 3H, CH3Cq of the other
stereoisomer couple); 1.28–1.17 (m, 12H, 3H of
CH3CH2 and 3H of CH3CH for each stereoisomer cou-
ple, respectively). 13C NMR (CDCl3, d): 172.55, 153.82,
129.48, 128.13, 121.27, 121.18, 85.80, 72.74, 72.47,
61.91, 16.85, 15.47, 14.27. GC–MS (70eV) (m/z) (rel
int.) 274 [M(37Cl)+, 3], 272 [M(35Cl)+, 8], 228 (23), 199
(13), 157 (11), 155 (34), 130 (34), 129 (13), 128 (100),
111 (9), 99 (19), 43 (36). Anal. Calcd for C13H17ClO4:
C, 57.35; H, 6.25. Found: C, 57.36; H, 6.27.
5.10. Synthesis of ethyl 2-(4-chlorophenoxy)-3-methoxy-
2-butenoate 4
To a solution of 1a (1g, 3.91mmol) in dry N,N-dimeth-
ylformamide (4mL) kept at room temperature under an
Table 6. Medium composition of ethyl 2-(4-chlorophenoxy)-3-acet-
oxy-2-butenoate 3 reduction reaction by using different procedures
Procedurea
BakerÕs yeast
(g/L)
Substrate
(g/L)
EtOH
(mL)
Reaction
time
5.9. Synthesis of ethyl 2-(4-chlorophenoxy)-3-acetoxy-2-
butenoate 3
B
150
400
3.5
5
—
12.5
2days
20h
D
a For procedures A, B, C and D, see also Section 5.1.
Acetic anhydride (48mL) was added dropwise to a solu-
tion of 1a (2.6g, 10.1mmol) in pyridine (47mL). The
reaction mixture was stirred at room temperature, while
monitoring the reaction progress by GC. After 15h, ice
was added. The mixture was extracted three times with
ethyl acetate. The organic layer was washed three times
with 1M HCl, three times with a saturated solution of
NaHCO3 and once with water. The organic extracts
were dried over anhydrous Na2SO4 and the solvent
evaporated under reduced pressure. The product was
isolated by chromatography (mobile phase: petroleum
Table 7. Medium composition of ethyl 2-(4-chlorophenoxy)-3-meth-
oxy-2-butenoate 4 reduction reaction by using different procedures
Procedurea
BakerÕs yeast
(g/L)
Substrate
(g/L)
EtOH
(mL)
Reaction
time
D
800
5
12.5
6days
a For procedures A, B, C and D, see also Section 5.1.

M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
3509
N2 atmosphere, Cs2CO3 (1.273g, 3.91mmol) was added.
CH3I (2.5mL, 39.06mmol) in dry N,N-dimethylform-
amide (8mL) was then added dropwise. The solution
was stirred at room temperature. Reaction progress
was monitored by TLC (mobile phase: petroleum
ether/ethyl ether = 8:2). After 22h, ethyl ether was
added and the mixture obtained, washed three times
with saturated Na2S2O3 and three times with saturated
NaHCO3. The extracts were dried over anhydrous
Na2SO4 and the solvent evaporated under reduced pres-
sure. The product was isolated by chromatography (mo-
bile phase: petroleum ether/ethyl ether = 8:2) as a
colourless oil. 5% yield (Table 7). IR (neat): 3050,
2922, 2856, 1706, 1627, 1593, 1486, 1456, 1381, 1273,
(I > 2r(I)): 0.0420/0.0992, Flack parameter = À0.04 (6),
À3
˚
largest diff. peak/hole: 0.430/À0.501eA
.
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, dellÕUniversita e della Ric-
erca (MIUR, Rome). Thanks are also due to the Univer-
sity of Bari and CNR-Istituto di Chimica dei Composti
OrganoMetallici-ICCOM/Sezione di Bari (Italy).
1223, 1137, 1098, 1006, 826cmÀ1 1H NMR (CDCl3,
.
References
d): 7.22–7.19 (m, 2H, aromatic protons); 6.85–6.82 (m,
2H, aromatic protons); 4.15–4.08 (q, J = 7.14Hz, 2H,
OCH2CH3); 3.75 (s, 3H, OCH3); 2.50 (s, 3H, CH3C@C);
1.15–1.10 (t, J = 7.14Hz, 3H, OCH2CH3). 13C NMR
(CDCl3, d): 165.28 (1C, COCH3); 160.65 (1C, CO);
157.11 (1C, CAr); 129.45 (2C, CAr); 126.63 (1C, CAr);
120.55 (1C, CCOOEt); 116.30 (2C, CAr); 60.88 (1C,
OCH2CH3); 56.37 (1C, OCH3); 14.59 (1C, CH3CH2O);
14.34 (1C, CH3CHOCH3). GC–MS (70eV) (m/z) (rel
int.) 272 [M(37Cl)+, 34], 270 [M(35Cl)+, 100], 225 (17),
199 (11), 197 (32), 175 (33), 169 (18), 167 (11), 162
(43), 161 (10), 147 (15), 141 (14), 139 (45), 137 (28),
125 (14), 115 (27), 113 (12), 111 (32), 103 (12), 99 (11),
85 (11), 75 (38), 57 (21), 43 (70).
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16. Di Nunno, L.; Franchini, C.; Scilimati, A.; Sinicropi, M.
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18. Roberts, S. M. Biocatalysts for Fine Chemicals Synthesis;
John Wiley & Sons: Chichester, 1999.
To establish the absolute configuration at C (7) and C
(9) in an unambiguous manner, suitable crystals were
grown and subjected to single-crystal X-ray analysis,
using a Nonius Kappa CCD area detector diffractome-
ter equipped with a fine focus sealed graphite-monoch-
˚
romated Mo-Ka radiation (k = 0.71073A). Data for
ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hydroxy-3-phenyl-
propanoate were collected at 293(2)K. Data reduction
and cell refinement were carried out with the programs
DENZO32 and COLLECT.33 The structure was solved
by the direct methods procedure of SIR97,34 while the
refinement processes were carried out on a full matrix
least squares technique using SHELXL-97.35 Detailed
crystal data and geometrical parameters are deposited
in the Supporting Information (cif file).36 The asymmet-
ric unit of ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hyd-
roxy-3-phenylpropanoate with the atomic numbering
scheme is depicted in Figure 2.
Pertinent crystallographic data for ethyl (2R,3S)-
2-(4-chlorophenoxy)-3-hydroxy-3-phenylpropanoate:
C17H17ClO4, Mr = 320.76gcmÀ3
group: P21, a = 10.4419 (1), b = 5.5948 (1), c = 13.5736
,
monoclinic, space
3
19. Faber, K. Biotransformations In Organic Chemistry, 2nd
ed.; Springer: Berlin, 1995.
20. Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M.
Biotransformation in Preparative Organic Chemistry;
Academic: San Diego, 1989.
21. Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.;
Leitersdorf, E.; Fruchart, J. C. Circulation 1998, 98, 2088–
2093.
˚
˚
(4)A, b = 97.163 (1)ꢁ, Cell volume = 786.79 (4)A ,
Z = 2, T = 293 (2)K, l =
qc = 1.354gcmÀ3
0.258mmÀ1
range = 2.33ꢁ–30.01ꢁ, hkl indices
,
,
h
À146h614, À76k67, À196l618, reflections (meas-
ured) = 12123, reflections (unique) = 4426, reflections
(unique [Fo > 2r{jFoj}]): 3355, Rint = 0.032, 267 parame-
ters, R1/wR2 (all data): 0.0665/0.1129, R1/wR2

3510
M. G. Perrone et al. / Tetrahedron: Asymmetry 15 (2004) 3501–3510
22. Bettoni, G.; Ferorelli, S.; Loiodice, F.; Tangari, N.;
Tortorella, V.; Gasparrini, F.; Misiti, D.; Villani, C.
Chirality 1992, 4, 193–203.
23. Loiodice, F.; Longo, A.; Bianco, P.; Tortorella, V.
Tetrahedron: Asymmetry 1995, 6, 1001–1011.
32. Otwinowski, Z.; Minor, W. In Methods in Enzymology,
Macromolecular Crystallography. Part A; Carter, C. W.,
Sweet, R. M., Eds.; Academic Press; Southwestern Med-
ical Centre at Dallas, HKL DENZO and Scalepack: USA,
1997; Vol. 276, p 307.
24. Chiaia Noya, F.; Ferorelli, S.; Franchini, C.; Scilimati, A.;
Sinicropi, M. S.; Tortorella, P. Il Farmaco 1996, 51, 293–
296.
25. Conte-Camerino, D.; Tortorella, V.; Ferrannini, E.; Bry-
ant, S. H. Arch. Toxicol. 1984, 7, 482–484.
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